Nucleic acids and proteins associated with sucrose accumulation in coffee

ABSTRACT

Disclosed herein are nucleic acid molecules isolated from coffee ( Coffea  spp.) comprising sequences that encodes various sucrose metabolizing enzymes, along with their encoded proteins. Specifically, sucrose synthase, sucrose phosphate synthase and sucrose phosphatase enzymes and their encoding polynucleotides from coffee are disclosed. Also disclosed are methods for using these polynucleotides for gene regulation and manipulation of the sugar profile of coffee plants, to influence flavor, aroma, and other features of coffee beans.

Divisional of U.S. application Ser. No. 11/990,549, filed Feb. 15, 2008,which is a U.S. National Phase of International Application No.PCT/US2006/032062, filed Aug. 16, 2006, which claims benefit of U.S.Provisional Application No. 60/709,043, filed Aug. 17, 2005, all ofwhich in their entirety are incorporated herein by reference.

This claims benefit of U.S. Provisional Application No. 60/709,043 filedAug. 17, 2005, the entire contents of which are incorporated byreference herein.

FIELD OF THE INVENTION

The present invention relates to the field of agriculturalbiotechnology. More particularly, the invention relates to enzymesparticipating in sucrose metabolism in plants, coffee in particular, andthe genes and nucleic acid sequences that encodes these enzymes, alongwith regulatory mechanisms that regulate the sucrose metabolism viathese enzymes.

BACKGROUND OF THE INVENTION

Various publications, including patents, published applications andscholarly articles, cited throughout the present specification areincorporated by reference herein, in their entireties. Citations notfully set forth within the specification may be found at the end of thespecification.

Sucrose plays an important role in the ultimate aroma and flavor that isdelivered by a coffee grain or bean. Sucrose is a major contributor tothe total free reducing sugars in coffee, and reducing sugars areimportant flavor precursors in coffee. During the roasting of coffeegrain, reducing sugars will react with amino group containing moleculesin a Maillard type reaction, which generates a significant number ofproducts with caramel, sweet and roast/burnt-type aromas and dark colorsthat are typically associated with coffee flavor (Russwurm, 1969;Holscher and Steinhart, 1995; Badoud, 2000). The highest quality Arabicagrain (Coffea Arabica) have been found to have appreciably higher levelsof sucrose (between 7.3 and 11.4%) than the lowest quality Robusta grain(Coffea canephora) (between 4 and 5%) (Russwurm, 1969; Illy and Viani,1995; Chahar et al., 2002; Badoud, 2000). Despite being significantlydegraded during roasting, sucrose still remains in the roasted grain atconcentrations of 0.4-2.8% dry weight (DW); thereby, contributingdirectly to coffee sweetness. A clear correlation exists between thelevel of sucrose in the grain and coffee flavor. Therefore, identifyingand isolating the major enzymes responsible for sucrose metabolism andthe underlying genetic basis for variations in sucrose metabolism willenable advances in the art of improving coffee quality.

Currently, there are no published reports on the genes or enzymesinvolved in sucrose metabolism in coffee. However, sucrose metabolismhas been studied in tomato Lycopersicon esculentum (a close relative ofcoffee, both are members of asterid I class), especially during tomatofruit development. An overview of the enzymes directly involved insucrose metabolism in tomato is shown in FIG. 1 (Nguyen-Quoc et al.,2001). The key reactions in this pathway are (1) the continuous rapiddegradation of sucrose in the cytosol by sucrose synthase (SuSy) andcytoplasmic invertase (I), (2) sucrose synthesis by SuSy orsucrose-phosphate synthase (SPS), (3) sucrose hydrolysis in the vacuoleor in the apoplast (region external to the plasma membrane, includingcell walls, xylem vessels, etc) by acid invertase (vacuolar or cell wallbound) and, (4) the rapid synthesis and breakdown of starch in theamyloplast.

As in other sink organs, the pattern of sucrose unloading is notconstant during tomato fruit development. At the early stages of fruitdevelopment, sucrose is unloaded intact from the phloem by the symplastpathway (direct connections between cells) and is not degraded to itscomposite hexoses during unloading. Both the expression and enzymeactivity of SuSy are highest at this stage and are directly correlatedwith sucrose unloading capacity from the phloem (phenomena also calledsink strength; Sun, et al., 1992; Zrenner et al., 1995). Later in fruitdevelopment, the symplastic connections are lost. Under these conditionsof unloading, sucrose is rapidly hydrolyzed outside the fruit cells bythe cell wall bound invertase and then the glucose and fructose productsare imported into the cells by hexose transporters. Sucrose issubsequently synthesized de novo in the cytoplasm by SuSy or SPS (FIG.1). SPS catalyses an essentially irreversible reaction in vivo due toits close association with the enzyme sucrose phosphate phosphatase(Echeverria et al., 1997). In parallel to the loss of the symplasticconnections, SuSy activity decreases, and eventually becomesundetectable in fruit at the onset of ripening (Robinson et al. 1998;Wang et al. 1993). Therefore, late in the development of tomato fruit,the SPS enzyme, in association with SP, appears as the major enzymes forsucrose synthesis.

During the past decade, evidence has increasingly indicated that SuSy isresponsible for the cleavage of newly imported sucrose, therebycontrolling the import capacity of the fruit (N'tchobo et al., 1999) andthe rate of starch synthesis. At the same time, SPS is now considered arate limiting enzyme in the pathway providing sucrose to plant storageorgans (roots, tubers and seeds) commonly referred to as sink. Together,this growing body of data strongly indicates that SuSy and SPS enzymesare important regulators of sucrose metabolism during tomato fruitdevelopment.

Alterations in carbon partitioning in plants, and most particularlyimprovement of sucrose levels in sink organs, have already beensuccessfully accomplished in several plants, the most extensive and mostencouraging results being obtained in tomato (Lycopersicon esculentum).Worrell and coworkers have made a set of constructions to test theeffects of increasing SPS levels. For the principle experiments, theyused a maize SPS cDNA under the control of the SSU promoter (Rubiscosmall subunit promoter) (Worrell, et al., 1991; Galtier et al. 1993;Foyer and Ferrario, 1994; Micallef, et al., 1995; Van Assche et al.,1999; Nguyen-Quoc et al., 1999). The total SPS activity in the leaves ofthe transformed plants was six times greater than that of the controls,while the total SPS activity in the mature fruit from the transformedplants was only twice than that of untransformed controls. Thisobservation suggests that, even with a strong constitutive promoter, thelevel of recombinant SPS was altered in a tissue specific manner.Interestingly, some results have also suggested that the maize SPSactivity was not under circadian control when this enzyme was expressedin tomato (Galtier et al., 1993). It should also be noted that SPSenzyme activity is negatively regulated at the post-translational levelby phosphorylation and the level of phosphorylation varies according tothe level of light and thus the light and dark phases of photosynthesis(Sugden et al., 1999; Jones et Ort, 1997). Therefore, the latter resultsuggests that the increase of SPS activity in the transgenic plants wasboth due to an over-expression of the protein and to the unregulatedactivity of the transfected maize SPS enzyme (i.e., the regulation byphosphorylation was perturbed). The increase in SPS activity wasaccompanied by a significant increase (25%) in total overall SuSyactivity in 20 day old tomato fruit. The SuSy activity was measured withan assay in the direction of sucrose breakdown (Nguyen-Quoc et al.;1999). Fruit from these transgenic tomato lines showed higher sugarcontent (36% increase) compared to untransformed plants (Van Assche etal., 1999). Biochemical studies have also shown that the high levels ofthe corn SPS activity in the plants caused a modification ofcarbohydrate portioning in the tomato leaves with an increase ofsucrose/starch ratios and also a strong improvement in photosyntheticcapacity. The tomato plants appeared to tolerate the elevated levels ofSPS as there were no apparent detrimental growing effects. Other plantstransformed with the construct 35SCaMV-SPS (35 S Cauliflower MosaïcVirus) have three to five times more total SPS activity in leaves thanin wild-type plants but surprisingly tomato fruit obtained from theseparticular transformants did not show any increase in SPS activity(Laporte et al. 1997; Nguyen-Quoc et al. 1999). These results indicatethat the promoter selected to drive transgene expression could play animportant role.

There remains a need to determine the metabolism of sucrose in coffeeand the enzymes involved in the metabolism. There is also a need toidentify and isolate the genes that encode these enzymes in coffee,thereby providing genetic and biochemical tools for modifying sucroseproduction in coffee beans to manipulate the flavor and aroma of thecoffee.

SUMMARY OF THE INVENTION

One aspect of the invention features a nucleic acid molecule isolatedfrom coffee (Coffea spp.) comprising a coding sequence that encodes asucrose synthase, sucrose phosphate synthase or sucrose phosphatase. Inone embodiment, the coding sequence encodes a sucrose synthase having anamino acid sequence comprising at least one fragment of SEQ ID NO:8comprising residues 7-554 or 565-727. In another embodiment, the sucrosesynthase has an amino acid sequence greater than 89% identical to SEQ IDNO:8, and preferably comprises SEQ ID NO:8. In other embodiments, thepolynucleotide encoding the sucrose synthase has 90% or greater identityto the coding sequence set forth in SEQ ID NO: 1, and preferablycomprises SEQ ID NO:1.

In another embodiment, the coding sequence encodes a sucrose phosphatesynthase having an amino acid sequence comprising at least one fragmentof SEQ ID NO:9 comprising residues 168-439 or 467-644. In anotherembodiment, the sucrose phosphate synthase has an amino acid sequencegreater than 83% identical to SEQ ID NO:9, and preferably comprises SEQID NO:9. In other embodiments, the nucleic acid molecule encodingsucrose phosphate synthase has a coding sequence greater than 79%identical to the coding sequence set forth in SEQ ID NO: 2, andpreferably comprises SEQ ID NO:2.

In another embodiment, the coding sequence encodes a sucrose phosphatasehaving an amino acid sequence comprising residues 1-408 of SEQ ID NO:10.In another embodiment, the sucrose phosphatase has an amino acidsequence greater than 81% identical to SEQ ID NO:10, and preferablycomprises SEQ ID NO:10. In another embodiment, the nucleic acid moleculecomprises a coding sequence greater than 78% identical to the codingsequence set forth in SEQ ID NO:3, and preferably comprises SEQ ID NO:3.

In certain embodiments, the coding sequence of the nucleic acid moleculeis an open reading frame of a gene. In other embodiments, it is a mRNAmolecule produced by transcription of the gene, or a cDNA moleculeproduced by reverse transcription of the mRNA molecule.

Another aspect of the invention features an oligonucleotide between 8and 100 bases in length, which is complementary to a segment of one ofthe aforementioned nucleic acid molecules.

Another aspect of the invention features a vector comprising the codingsequence of the nucleic acid molecule described above. In someembodiments, the vector is an expression vector selected from the groupof vectors consisting of plasmid, phagemid, cosmid, baculovirus, bacmid,bacterial, yeast and viral vectors. In one embodiment, the codingsequence of the nucleic acid molecule is operably linked to aconstitutive promoter. In another embodiment, the coding sequence of thenucleic acid molecule is operably linked to an inducible promoter. Inanother embodiment, the coding sequence of the nucleic acid molecule isoperably linked to a tissue specific promoter, particularly a seedspecific promoter, and more specifically a coffee seed specificpromoter.

Another aspect of the invention features a host cell transformed withthe vector described above. In various embodiments, the host cell is aplant cell, bacterial cell, fungal cell, insect cell or mammalian cell.In specific embodiments, the host cell is a plant cell from a plant suchas coffee, tobacco, Arabidopsis, maize, wheat, rice, soybean barley,rye, oats, sorghum, alfalfa, clover, canola, safflower, sunflower,peanut, cacao, tomatillo, potato, pepper, eggplant, sugar beet, carrot,cucumber, lettuce, pea, aster, begonia, chrysanthemum, delphinium,zinnia, or turfgrasses.

In accordance with another aspect of the invention, a fertile plantproduced from the aforementioned transformed plant cell is provided.

Yet another aspect of the invention provides a method of modulatingflavor or aroma of coffee beans, comprising modulating production oractivity of one or more sucrose metabolizing enzymes within coffeeseeds. In one embodiment, the modulating comprises increasing productionor activity of the one or more sucrose metabolizing enzymes. This may beaccomplished increasing expression of one or more endogenous sucrosemetabolizing enzyme-encoding genes within the coffee seeds, which, incertain embodiments, is achieved by introducing a sucrose metabolizingenzyme-encoding transgene into the plant. In one embodiment, thetransgene encodes sucrose phosphate synthase. In a particularembodiment, the plant comprises more sucrose in its seeds than does anequivalent plant that does not contain the transgene. In anotherembodiment, the sucrose metabolizing enzyme is sucrose phosphatesynthase and is modified by removal of one or more phosphorylationsites, thereby increasing activity of the enzyme.

In another embodiment, the method of modulation comprises decreasingproduction or activity of the one or more sucrose metabolizing enzymes.In certain embodiments, this may be accomplished by introducing anucleic acid molecule into the coffee that inhibits the expression ofone or more of the sucrose metabolizing enzyme-encoding genes.

Other features and advantages of the present invention will beunderstood by reference to the drawings, detailed description andexamples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Model for sucrose metabolism in tomato fruit. Sucrose (S) isimported from phloem by a symplastic pathway or is hydrolyzed bycell-wall invertase. Glucose and fructose are imported into the cytosolby specific Sugar Transporter Proteins. In cytosol, sucrose is degradedby sucrose synthase (SS) and its re-synthesis is catalysed by sucrosephosphate synthase (SPS) associated with sucrose phosphatase (SP) or SS.Sucrose can be exported in vacuole and hydrolysed by vacuolar invertase.UDP-glucose after modifications can be used for starch synthesis inchromoplast. Abbreviations: G, glucose; F, fructose; F6-P, fructose6-phosphate; UDP-G, UDP-glucose; G6-P, glucose 6-phosphate; S6-P,sucrose 6-phosphate; I, invertase; SP, sucrose phosphatase; SPS sucrosephosphate synthase.

FIG. 2. Protein sequence alignment of CcSS2 with other SuSy proteinssequences. CcSS2 protein is aligned with other sucrose synthase proteinsavailable in the NCBI database was done using the CLUSTAL W program inthe MegAlign software (Lasergene package, DNASTAR). Amino acidsunderlined in red are different from CcSS2 protein. GenBank accessionnumbers are AY205084 for potato SuSyST2 (Solanum tuberosum) (SEQ IDNO.:11), AJ537575 for potato SuSyST4 (SEQ ID NO.:12) and AJ011535 fortomato SuSyLE2 (Lycopersicon esculentum) (SEQ ID NO.:13).

FIG. 3. Schematic representation of CcSPS1 gene from C. canephora. TheSPS-C1 fragment has been amplified by PCR from BP-409 genomic DNA usingthe degenerate primers SPS-3 and SPS-4. Successive genome walkingexperiments subsequently permitted the amplification of the overlappingfragments for the 5′ and 3′ flanking regions of SPS-C1. Alignment of theresulting genomic clones (C1-12, C1-GW4-23, C1-62 and C1-GW1-11) haslead to the complete sequence of CcSPS1 gene. The putative proteincoding region has been localized by alignment with the closely relatedsucrose phosphate synthase protein SPSLE1 (accession number No.AAC24872) (SEQ ID NO.:14) from tomato (Lycopersicon esculentum). Theprotein-coding regions are shown in black. Triangles indicate theposition of the translation initiation start (ATG) and stop (TAG)codons.

FIG. 4. Protein sequence alignment of CcSPS1 with other SPS proteins.Alignment of protein encoded by the CcSPS1 cDNA with other SPS proteinsavailable in the NCBI database was done using the CLUSTAL W program inthe MegAlign software (Lasergene package, DNASTAR). Amino acids markedin red are different from the CcSPS1 protein. The other SPS proteins,with the associated accession number in parentheses, are as follows:SPSST (Solanum tuberosum, CAA51872) (SEQ ID NO.:15), SPSLE1(Lycopersicon esculentum, AAC24872) (SEQ ID NO.:14), SPSNT (Nicotianatabacum, AAF06792) (SEQ ID NO.:16), SPSLE2 (Lycopersicon esculentum,AAU29197) (SEQ ID NO.:17). The three sites for potential serylphosphorylation of CcSPS1 protein are indicated by an asterisk (Ser150,Ser221 and Ser415). A highly conserved sequence surrounding each serineis also shown.

FIG. 5. Protein sequence alignment of CcSP1 with other SP proteins.Alignment of CcSP1 protein with other SP proteins available in the NCBIdatabase was done using the CLUSTAL W program in the MegAlign software(Lasergene package, DNASTAR). Amino acids underlined in red aredifferent from CcSP1 protein. GenBank accession numbers are NP_(—)973609for Arabidopsis SPAT1 (Arabidopsis thaliana) (SEQ ID NO.:18) andAAO33160 for tomato SPLE (Lycopersicon esculentum) (SEQ ID NO.:19).

FIG. 6. Changes in activity of SPS and SuSy activity and concentrationsof sucrose, glucose and fructose in whole grains (separated frompericarp and locules) during (A) FRT05 C. canephara and (B) CCCA12 C.arabica coffee grain maturation. Coffee cherries at four differentmaturation stages characterized by size and color were used, i.e., SG(small green), LG (large green), Y (yellow) and R (red). Concentrationsof sucrose, glucose and fructose in the coffee grain were measured insamples harvested in parallel to those used for the assays of SPS andSuSy activity. Sugar concentration is expressed in g/100 g DW whileenzymatic activities are expressed in μmoles/h/mg protein.

FIG. 7. Tissue-specific mRNA expression profiles of CcSS2, CcSPS1 andCcSP1 in C. canephora (Robusta, BP409) and C. arabica (Arabica, T2308)using real-time RT-PCR. Total RNA was isolated from root, flower, leafand coffee beans harvested at four different maturation stages i.e.Small-Green (SG), Large-Green (LG), Yellow (Y) and Red (R). For eachmaturation stage, coffee cherries have been separated from pericarp (P)and grains (G). Total RNA was reverse transcribed and subjected toreal-time PCR using TaqMan-MGB probes. Relative amounts were calculatedand normalized with respect to rp139 transcript levels. Data shownrepresent mean values obtained from three amplification reactions andthe error bars indicate the SD of the mean.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Definitions

Various terms relating to the biological molecules and other aspects ofthe present invention are used through the specification and claims. Theterms are presumed to have their customary meaning in the field ofmolecular biology and biochemistry unless they are specifically definedotherwise herein.

The term “sucrose metabolizing enzyme” refers to enzymes in plants thatprimarily function to accumulate sucrose or degrade sucrose within theplant and include, for example, sucrose synthase (SuSy), sucrosephosphate synthase (SPS) and sucrose phosphatase (SP). Together, thedifferent sucrose metabolizing enzymes operate to control the metabolismof sucrose as needed by the plant for either storage or for energyneeds.

“Isolated” means altered “by the hand of man” from the natural state. Ifa composition or substance occurs in nature, it has been “isolated” ifit has been changed or removed from its original environment, or both.For example, a polynucleotide or a polypeptide naturally present in aliving plant or animal is not “isolated,” but the same polynucleotide orpolypeptide separated from the coexisting materials of its natural stateis “isolated”, as the term is employed herein.

“Polynucleotide”, also referred to as “nucleic acid molecule”, generallyrefers to any polyribonucleotide or polydeoxyribonucleotide, which maybe unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides”include, without limitation single- and double-stranded DNA, DNA that isa mixture of single- and double-stranded regions, single- anddouble-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or a mixtureof single- and double-stranded regions. In addition, “polynucleotide”refers to triple-stranded regions comprising RNA or DNA or both RNA andDNA. The term polynucleotide also includes DNAs or RNAs containing oneor more modified bases and DNAs or RNAs with backbones modified forstability or for other reasons. “Modified” bases include, for example,tritylated bases and unusual bases such as inosine. A variety ofmodifications can be made to DNA and RNA; thus, “polynucleotide”embraces chemically, enzymatically or metabolically modified forms ofpolynucleotides as typically found in nature, as well as the chemicalforms of DNA and RNA characteristic of viruses and cells.“Polynucleotide” also embraces relatively short polynucleotides, oftenreferred to as oligonucleotides.

“Polypeptide” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. “Polypeptide” refers to both shortchains, commonly referred to as peptides, oligopeptides or oligomers,and to longer chains, generally referred to as proteins. Polypeptidesmay contain amino acids other than the 20 gene-encoded amino acids.“Polypeptides” include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from natural posttranslational processesor may be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination. See, for instance, Proteins—Structure and MolecularProperties, 2nd Ed., T. E. Creighton, W, H. Freeman and Company, NewYork, 1993 and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in Posttranslational CovalentModification of Proteins, B. C. Johnson, Ed., Academic Press, New York,1983; Seifter et al., “Analysis for Protein Modifications and NonproteinCofactors”, Meth Enzymol (1990) 182:626-646 and Rattan et al., “ProteinSynthesis: Posttranslational Modifications and Aging”, Ann NY Acad Sci(1992) 663:48-62.

“Variant” as the term is used herein, is a polynucleotide or polypeptidethat differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, or deletions in any combination. A substitutedor inserted amino acid residue may or may not be one encoded by thegenetic code. A variant of a polynucleotide or polypeptide may benaturally occurring, such as an allelic variant, or it may be a variantthat is not known to occur naturally. Non-naturally occurring variantsof polynucleotides and polypeptides may be made by mutagenesistechniques or by direct synthesis.

In reference to mutant plants, the terms “null mutant” or“loss-of-function mutant” are used to designate an organism or genomicDNA sequence with a mutation that causes a gene product to benon-functional or largely absent. Such mutations may occur in the codingand/or regulatory regions of the gene, and may be changes of individualresidues, or insertions or deletions of regions of nucleic acids. Thesemutations may also occur in the coding and/or regulatory regions ofother genes which may regulate or control a gene and/or encoded protein,so as to cause the protein to be non-functional or largely absent.

The term “substantially the same” refers to nucleic acid or amino acidsequences having sequence variations that do not materially affect thenature of the protein (i.e. the structure, stability characteristics,substrate specificity and/or biological activity of the protein). Withparticular reference to nucleic acid sequences, the term “substantiallythe same” is intended to refer to the coding region and to conservedsequences governing expression, and refers primarily to degeneratecodons encoding the same amino acid, or alternate codons encodingconservative substitute amino acids in the encoded polypeptide. Withreference to amino acid sequences, the term “substantially the same”refers generally to conservative substitutions and/or variations inregions of the polypeptide not involved in determination of structure orfunction.

The terms “percent identical” and “percent similar” are also used hereinin comparisons among amino acid and nucleic acid sequences. Whenreferring to amino acid sequences, “identity” or “percent identical”refers to the percent of the amino acids of the subject amino acidsequence that have been matched to identical amino acids in the comparedamino acid sequence by a sequence analysis program. “Percent similar”refers to the percent of the amino acids of the subject amino acidsequence that have been matched to identical or conserved amino acids.Conserved amino acids are those which differ in structure but aresimilar in physical properties such that the exchange of one for anotherwould not appreciably change the tertiary structure of the resultingprotein. Conservative substitutions are defined in Taylor (1986, J.Theor. Biol. 119:205). When referring to nucleic acid molecules,“percent identical” refers to the percent of the nucleotides of thesubject nucleic acid sequence that have been matched to identicalnucleotides by a sequence analysis program.

“Identity” and “similarity” can be readily calculated by known methods.Nucleic acid sequences and amino acid sequences can be compared usingcomputer programs that align the similar sequences of the nucleic oramino acids and thus define the differences. In preferred methodologies,the BLAST programs (NCBI) and parameters used therein are employed, andthe DNAstar system (Madison, Wis.) is used to align sequence fragmentsof genomic DNA sequences. However, equivalent alignments andsimilarity/identity assessments can be obtained through the use of anystandard alignment software. For instance, the GCG Wisconsin Packageversion 9.1, available from the Genetics Computer Group in Madison,Wis., and the default parameters used (gap creation penalty=12, gapextension penalty=4) by that program may also be used to comparesequence identity and similarity.

“Antibodies” as used herein includes polyclonal and monoclonalantibodies, chimeric, single chain, and humanized antibodies, as well asantibody fragments (e.g., Fab, Fab′, F(ab′)₂ and F_(v)), including theproducts of a Fab or other immunoglobulin expression library. Withrespect to antibodies, the term, “immunologically specific” or“specific” refers to antibodies that bind to one or more epitopes of aprotein of interest, but which do not substantially recognize and bindother molecules in a sample containing a mixed population of antigenicbiological molecules. Screening assays to determine binding specificityof an antibody are well known and routinely practiced in the art. For acomprehensive discussion of such assays, see Harlow et al. (Eds.),ANTIBODIES A LABORATORY MANUAL; Cold Spring Harbor Laboratory; ColdSpring Harbor, N.Y. (1988), Chapter 6.

The term “substantially pure” refers to a preparation comprising atleast 50-60% by weight the compound of interest (e.g., nucleic acid,oligonucleotide, protein, etc.). More preferably, the preparationcomprises at least 75% by weight, and most preferably 90-99% by weight,the compound of interest. Purity is measured by methods appropriate forthe compound of interest (e.g. chromatographic methods, agarose orpolyacrylamide gel electrophoresis, HPLC analysis, and the like).

With respect to single-stranded nucleic acid molecules, the term“specifically hybridizing” refers to the association between twosingle-stranded nucleic acid molecules of sufficiently complementarysequence to permit such hybridization under pre-determined conditionsgenerally used in the art (sometimes termed “substantiallycomplementary”). In particular, the term refers to hybridization of anoligonucleotide with a substantially complementary sequence containedwithin a single-stranded DNA or RNA molecule, to the substantialexclusion of hybridization of the oligonucleotide with single-strandednucleic acids of non-complementary sequence.

A “coding sequence” or “coding region” refers to a nucleic acid moleculehaving sequence information necessary to produce a gene product, whenthe sequence is expressed. The coding sequence may comprise untranslatedsequences (e.g., introns) within translated regions, or may lack suchintervening untranslated sequences (e.g., as in cDNA).

“Intron” refers to polynucleotide sequences in a nucleic acid that donot code information related to protein synthesis. Such sequences aretranscribed into mRNA, but are removed before translation of the mRNAinto a protein.

The term “operably linked” or “operably inserted” means that theregulatory sequences necessary for expression of the coding sequence areplaced in a nucleic acid molecule in the appropriate positions relativeto the coding sequence so as to enable expression of the codingsequence. By way of example, a promoter is operably linked with a codingsequence when the promoter is capable of controlling the transcriptionor expression of that coding sequence. Coding sequences can be operablylinked to promoters or regulatory sequences in a sense or antisenseorientation. The term “operably linked” is sometimes applied to thearrangement of other transcription control elements (e.g. enhancers) inan expression vector.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, polyadenylation signals,terminators, and the like, that provide for the expression of a codingsequence in a host cell.

The terms “promoter”, “promoter region” or “promoter sequence” refergenerally to transcriptional regulatory regions of a gene, which may befound at the 5′ or 3′ side of the coding region, or within the codingregion, or within introns. Typically, a promoter is a DNA regulatoryregion capable of binding RNA polymerase in a cell and initiatingtranscription of a downstream (3′ direction) coding sequence. Thetypical 5′ promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence is a transcription initiation site (conveniently defined bymapping with nuclease S1), as well as protein binding domains (consensussequences) responsible for the binding of RNA polymerase.

A “vector” is a replicon, such as plasmid, phage, cosmid, or virus towhich another nucleic acid segment may be operably inserted so as tobring about the replication or expression of the segment.

The term “nucleic acid construct” or “DNA construct” is sometimes usedto refer to a coding sequence or sequences operably linked toappropriate regulatory sequences and inserted into a vector fortransforming a cell. This term may be used interchangeably with the term“transforming DNA” or “transgene”. Such a nucleic acid construct maycontain a coding sequence for a gene product of interest, along with aselectable marker gene and/or a reporter gene.

A “marker gene” or “selectable marker gene” is a gene whose encoded geneproduct confers a feature that enables a cell containing the gene to beselected from among cells not containing the gene. Vectors used forgenetic engineering typically contain one or more selectable markergenes. Types of selectable marker genes include (1) antibioticresistance genes, (2) herbicide tolerance or resistance genes, and (3)metabolic or auxotrophic marker genes that enable transformed cells tosynthesize an essential component, usually an amino acid, which thecells cannot otherwise produce.

A “reporter gene” is also a type of marker gene. It typically encodes agene product that is assayable or detectable by standard laboratorymeans (e.g., enzymatic activity, fluorescence).

The term “express,” “expressed,” or “expression” of a gene refers to thebiosynthesis of a gene product. The process involves transcription ofthe gene into mRNA and then translation of the mRNA into one or morepolypeptides, and encompasses all naturally occurring post-translationalmodifications.

“Endogenous” refers to any constituent, for example, a gene or nucleicacid, or polypeptide, that can be found naturally within the specifiedorganism.

A “heterologous” region of a nucleic acid construct is an identifiablesegment (or segments) of the nucleic acid molecule within a largermolecule that is not found in association with the larger molecule innature. Thus, when the heterologous region comprises a gene, the genewill usually be flanked by DNA that does not flank the genomic DNA inthe genome of the source organism. In another example, a heterologousregion is a construct where the coding sequence itself is not found innature (e.g., a cDNA where the genomic coding sequence contains introns,or synthetic sequences having codons different than the native gene).Allelic variations or naturally-occurring mutational events do not giverise to a heterologous region of DNA as defined herein. The term “DNAconstruct”, as defined above, is also used to refer to a heterologousregion, particularly one constructed for use in transformation of acell.

A cell has been “transformed” or “transfected” by exogenous orheterologous DNA when such DNA has been introduced inside the cell. Thetransforming DNA may or may not be integrated (covalently linked) intothe genome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA. A “clone” is a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

“Grain,” “seed,” or “bean,” refers to a flowering plant's unit ofreproduction, capable of developing into another such plant. As usedherein, especially with respect to coffee plants, the terms are usedsynonymously and interchangeably.

As used herein, the term “plant” includes reference to whole plants,plant organs (e.g., leaves, stems, shoots, roots), seeds, pollen, plantcells, plant cell organelles, and progeny thereof. Parts of transgenicplants are to be understood within the scope of the invention tocomprise, for example, plant cells, protoplasts, tissues, callus,embryos as well as flowers, stems, seeds, pollen, fruits, leaves, orroots originating in transgenic plants or their progeny.

Description

Sucrose is a major contributor of free reducing sugars involved in theMaillard reaction that occurs during the roasting of coffee grain.Therefore, it is widely believed to be an important flavor precursormolecule in the green coffee grain. Consistent with this idea, thehighest quality Arabica grains have appreciably higher levels of sucrose(between 7.3 and 11.4%) than the lowest quality Robusta grains (between4 and 5%). Also, sucrose, while being significantly degraded duringroasting, can remain in the roasted grain at concentrations of 0.4-2.8%dry weight (DW) and so participates directly in coffee's sweetness.Because of the clear correlation between the level of sucrose in thegrain and coffee flavor, the ability to understand and manipulate theunderlying genetic basis for variations in sucrose metabolism and carbonpartitioning in coffee grain is important.

Key enzymes involved in sucrose metabolism have been characterized inmodel organisms (e.g., tomato, potato, Arabidopsis). In accordance withthe present invention, protein sequences of these enzymes have been usedto perform similarity searches in Coffea canephora cDNA libraries andEST databases using the tBLASTn algorithm, as described in greaterdetail in the examples. cDNA encoding sucrose synthase (CcSS2) (SEQ IDNO:1), sucrose phosphate synthase (CcSPS1) (SEQ ID NO:2), sucrosephosphatase (CcSP1) (SEQ ID NO:3) were identified and characterized inC. canephora. A partial cDNA sequence of CcSPS1 has also beenidentified, and is referred to herein as SEQ ID NO:7.

Using degenerate primers, a partial genomic clone of a sucrose phosphatesynthase CcSPS1-encoding gene (SEQ ID NO:4) has been isolated from C.canephora. A second gene was also isolated, and referred to herein asCcSPS2 (SEQ ID NO:5). Confirmation of expression was performed withCcSPS1 by sequencing the single PCR fragment obtained after RT-PCR. Acomplete genomic clone of a CcSPS1-encoding gene was identified and isreferred to herein as SEQ ID NO:6.

Eleven single nucleotide polymorphisms (SNPs) have been identified inthe CcSPS1 full length genomic clone. It is expected that these SNPs andother sequence markers will be useful for placing the CcSPS1 gene on aC. canephora genetic map.

The study of SuSy and SPS activity during grain development in a varietyof Arabica (C. Arabica CCCA12) and Robusta (C. canephora FRT05) grainhas shown that, although the Robusta variety was characterized by astronger sink strength (correlated to higher SuSy activity), the Arabicavariety accumulated 30% more sucrose in mature beans than did theRobusta variety. Additionally, while SPS activity fluctuated during theRobusta grain development, the SPS activity in Arabica rose rapidly andremained high up to grain maturity. It was found that CcSS2 and CcSPS1mRNA accumulation was highly correlated with enzymatic activity. Thedata obtained in accordance with the invention described herein stronglyindicate that SPS activity is the limiting step for re-synthesis ofsucrose during final step of coffee grain maturation. In the perspectiveof improving the quality of Robusta and other coffee grain, selection ofvarieties with high SPS activity, or manipulation of plants to increaseSPS production or activity, are expected to be an important route forincreasing the final sucrose concentration in mature coffee bean.

Thus, one aspect of the present invention relates to nucleic acidmolecules from coffee that encode a number of sucrose metabolizingenzymes, including sucrose synthase (SuSy), exemplified by SEQ ID NO:1,sucrose phosphate synthase (SPS), exemplified by SEQ ID NO:2 (and thepartial sequence of SEQ ID NO:7), and the open reading frame of SEQ IDNO:6 (and by the partial open reading frames of SEQ ID NOS: 4 and 5described herein), and sucrose phosphatase (SP), exemplified by SEQ IDNO:3. Other aspects of the invention relate to the proteins produced byexpression of these nucleic acid molecules and their uses. The deducedamino acid sequences of the proteins produced by expression of SEQ IDNOS: 1, 2 or 3 are set forth herein as SEQ NO:8 (SuSy), SEQ ID NO:9(SPS) and SEQ ID NO:10 (SP). The predicted molecular masses of theseproteins are 92.6 kDa (SuSy), 117 kDa (SPS) and 46.7 kDa (SP). Stillother aspects of the invention relate to uses of the nucleic acidmolecules and encoded polypeptides in plant breeding and in geneticmanipulation of plants, and ultimately in the manipulation of coffeeflavor, aroma and other qualities.

Although polynucleotides encoding sucrose metabolizing enzymes fromCoffea canephora are described and exemplified herein, this invention isintended to encompass nucleic acids and encoded proteins from otherCoffea species that are sufficiently similar to be used interchangeablywith the C. canephora polynucleotides and proteins for the purposesdescribed below. Accordingly, when the terms “sucrose synthase,”“sucrose phosphate synthase,” and “sucrose phosphatase” are used herein,they are intended to encompass all Coffea sucrose synthases, sucrosephosphate synthases, and sucrose phosphatases having the generalphysical, biochemical and functional features described herein, andpolynucleotides encoding them.

Considered in terms of their sequences, sucrose metabolizingenzyme-encoding polynucleotides of the invention include allelicvariants and natural mutants of any of SEQ ID NOS: 1-7, which are likelyto be found in different varieties of C. canephora, and homologs of SEQID NOS: 1-7 likely to be found in different coffee species. Because suchvariants and homologs are expected to possess certain differences innucleotide and amino acid sequence, this invention provides isolatedsucrose metabolizing enzyme-encoding nucleic acid molecules that encoderespective polypeptides having at least about 80% (and, with increasingorder of preference, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99%) identity with the codingregions of any one of SEQ ID NOS: 8-10 and comprises a nucleotidesequence having equivalent ranges of identity to the pertinent portionsof any one of SEQ ID NOS: 1-7, respectively. Because of the naturalsequence variation likely to exist among sucrose metabolizing enzymes,and the genes encoding them in different coffee varieties and species,one skilled in the art would expect to find this level of variation,while still maintaining the unique properties of the polypeptides andpolynucleotides of the present invention. Such an expectation is due inpart to the degeneracy of the genetic code, as well as to the knownevolutionary success of conservative amino acid sequence variations,which do not appreciably alter the nature of the encoded protein.Accordingly, such variants and homologs are considered substantially thesame as one another and are included within the scope of the presentinvention.

The following sections set forth the general procedures involved inpracticing the present invention. To the extent that specific materialsare mentioned, it is merely for the purpose of illustration, and is notintended to limit the invention. Unless otherwise specified, generalbiochemical and molecular biological procedures, such as those set forthin Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory(1989) or Ausubel et al. (eds), Current Protocols in Molecular Biology,John Wiley & Sons (2005) are used.

Nucleic Acid Molecules, Proteins and Antibodies:

Nucleic acid molecules of the invention may be prepared by two generalmethods: (1) they may be synthesized from appropriate nucleotidetriphosphates, or (2) they may be isolated from biological sources. Bothmethods utilize protocols well known in the art.

The availability of nucleotide sequence information, such as the cDNAhaving SEQ ID NOS: 1, 2 or 3, (or the open reading frame of SEQ ID NO:6)enables preparation of an isolated nucleic acid molecule of theinvention by oligonucleotide synthesis. Synthetic oligonucleotides maybe prepared by the phosphoramidite method employed in the AppliedBiosystems 38A DNA Synthesizer or similar devices. The resultantconstruct may be purified according to methods known in the art, such ashigh performance liquid chromatography (HPLC). Long, double-strandedpolynucleotides, such as a DNA molecule of the present invention, mustbe synthesized in stages, due to the size limitations inherent incurrent oligonucleotide synthetic methods. Thus, for example, a longdouble-stranded molecule may be synthesized as several smaller segmentsof appropriate complementarity. Complementary segments thus produced maybe annealed such that each segment possesses appropriate cohesivetermini for attachment of an adjacent segment. Adjacent segments may beligated by annealing cohesive termini in the presence of DNA ligase toconstruct an entire long double-stranded molecule. A synthetic DNAmolecule so constructed may then be cloned and amplified in anappropriate vector.

In accordance with the present invention, nucleic acids having theappropriate level sequence homology with part or all of the codingand/or regulatory regions of sucrose metabolizing enzyme-encodingpolynucleotides may be identified by using hybridization and washingconditions of appropriate stringency. It will be appreciated by thoseskilled in the art that the aforementioned strategy, when applied togenomic sequences, will, in addition to enabling isolation of sucrosemetabolizing enzyme-coding sequences, also enable isolation of promotersand other gene regulatory sequences associated with sucrose metabolizingenzyme genes, even though the regulatory sequences themselves may notshare sufficient homology to enable suitable hybridization.

As a typical illustration, hybridizations may be performed, according tothe method of Sambrook et al., using a hybridization solutioncomprising: 5×SSC, 5×Denhardt's reagent, 1.0% SDS, 100 μg/ml denatured,fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50%formamide, Hybridization is carried out at 37-42° C. for at least sixhours. Following hybridization, filters are washed as follows: (1) 5minutes at room temperature in 2×SSC and 1% SDS; (2) 15 minutes at roomtemperature in 2×SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37° C. in2×SSC and 0.1% SDS; (4) 2 hours at 45-55° C. in 2×SSC and 0.1% SDS,changing the solution every 30 minutes.

One common formula for calculating the stringency conditions required toachieve hybridization between nucleic acid molecules of a specifiedsequence homology (Sambrook et al., 1989):T_(m)=81.5° C.+16.6 Log [Na+]+0.41(% G+C)−0.63(% formamide)−600/#bp induplex

As an illustration of the above formula, using [Na+]=[0.368] and 50%formamide, with GC content of 42% and an average probe size of 200bases, the Tm is 57° C. The Tm of a DNA duplex decreases by 1-1.5° C.with every 1% decrease in homology. Thus, targets with greater thanabout 75% sequence identity would be observed using a hybridizationtemperature of 42° C. In one embodiment, the hybridization is at 37° C.and the final wash is at 42° C.; in another embodiment the hybridizationis at 42° C. and the final wash is at 50° C.; and in yet anotherembodiment the hybridization is at 42° C. and final wash is at 65° C.,with the above hybridization and wash solutions. Conditions of highstringency include hybridization at 42° C. in the above hybridizationsolution and a final wash at 65° C. in 0, 1×SSC and 0.1% SDS for 10minutes.

Nucleic acids of the present invention may be maintained as DNA in anyconvenient cloning vector. In a preferred embodiment, clones aremaintained in plasmid cloning/expression vector, such as pGEM-T (PromegaBiotech, Madison, Wis.), pBluescript (Stratagene, La Jolla, Calif.),pCR4-TOPO (Invitrogen, Carlsbad, Calif.) or pET28a+ (Novagen, Madison,Wis.), all of which can be propagated in a suitable E. coli host cell.

Nucleic acid molecules of the invention include cDNA, genomic DNA, RNA,and fragments thereof which may be single-, double-, or eventriple-stranded. Thus, this invention provides oligonucleotides (senseor antisense strands of DNA or RNA) having sequences capable ofhybridizing with at least one sequence of a nucleic acid molecule of thepresent invention. Such oligonucleotides are useful as probes fordetecting sucrose metabolizing enzyme encoding genes or mRNA in testsamples of plant tissue, e.g., by PCR amplification, or for the positiveor negative regulation of expression of sucrose metabolizingenzyme-encoding genes at or before translation of the mRNA intoproteins. Methods in which sucrose metabolizing enzyme-encodingoligonucleotides or polynucleotides may be utilized as probes for suchassays include, but are not limited to: (1) in situ hybridization; (2)Southern hybridization (3) northern hybridization; and (4) assortedamplification reactions such as polymerase chain reactions (PCR,including RT-PCR) and ligase chain reaction (LCR).

Polypeptides encoded by nucleic acids of the invention may be preparedin a variety of ways, according to known methods. If produced in situthe polypeptides may be purified from appropriate sources, e.g., seeds,pericarps, or other plant parts.

Alternatively, the availability of isolated nucleic acid moleculesencoding the SuSy, SPS or SP polypeptides enables production of theproteins using in vitro expression methods known in the art. Forexample, a cDNA or gene may be cloned into an appropriate in vitrotranscription vector, such as pSP64 or pSP65 for in vitro transcription,followed by cell-free translation in a suitable cell-free translationsystem, such as wheat germ or rabbit reticulocytes. In vitrotranscription and translation systems are commercially available, e.g.,from Promega Biotech, Madison, Wis., BRL, Rockville, Md. or Invitrogen,Carlsbad, Calif.

According to a preferred embodiment, larger quantities of sucrosemetabolizing enzymes may be produced by expression in a suitableprocaryotic or eucaryotic system. For example, part or all of a DNAmolecule, such as the cDNAs having SEQ ID NOS: 1, 2 or 3, may beinserted into a plasmid vector adapted for expression in a bacterialcell (such as E. coli) or a yeast cell (such as Saccharomycescerevisiae), or into a baculovirus vector for expression in an insectcell. Such vectors comprise the regulatory elements necessary forexpression of the DNA in the host cell, positioned in such a manner asto permit expression of the DNA in the host cell. Such regulatoryelements required for expression include promoter sequences,transcription initiation sequences and, optionally, enhancer sequences.

The sucrose metabolizing enzymes produced by gene expression in arecombinant procaryotic or eucyarotic system may be purified accordingto methods known in the art. In a preferred embodiment, a commerciallyavailable expression/secretion system can be used, whereby therecombinant protein is expressed and thereafter secreted from the hostcell, and, thereafter, purified from the surrounding medium. Analternative approach involves purifying the recombinant protein byaffinity separation, e.g., via immunological interaction with antibodiesthat bind specifically to the recombinant protein.

The sucrose metabolizing enzymes of the invention, prepared by theaforementioned methods, may be analyzed according to standardprocedures.

Sucrose metabolizing enzymes purified from coffee or recombinantlyproduced, may be used to generate polyclonal or monoclonal antibodies,antibody fragments or derivatives as defined herein, according to knownmethods. In addition to making antibodies to the entire recombinantprotein, if analyses of the proteins or Southern and cloning analyses(see below) indicate that the cloned genes belongs to a multigenefamily, then member-specific antibodies made to synthetic peptidescorresponding to nonconserved regions of the protein can be generated.

Kits comprising an antibody of the invention for any of the purposesdescribed herein are also included within the scope of the invention. Ingeneral, such a kit includes a control antigen for which the antibody isimmunospecific.

Vectors, Cells, Tissues and Plants:

Also featured in accordance with the present invention are vectors andkits for producing transgenic host cells that contain a sucrosemetabolizing enzyme-encoding polynucleotide or oligonucleotide, orhomolog, analog or variant thereof in a sense or antisense orientation,or reporter gene and other constructs under control of sucrosemetabolizing enzyme-promoters and other regulatory sequences. Suitablehost cells include, but are not limited to, plant cells, bacterialcells, yeast and other fungal cells, insect cells and mammalian cells.Vectors for transforming a wide variety of these host cells are wellknown to those of skill in the art. They include, but are not limitedto, plasmids, cosmids, baculoviruses, bacmids, bacterial artificialchromosomes (BACs), yeast artificial chromosomes (YACs), as well asother bacterial, yeast and viral vectors. Typically, kits for producingtransgenic host cells will contain one or more appropriate vectors andinstructions for producing the transgenic cells using the vector. Kitsmay further include one or more additional components, such as culturemedia for culturing the cells, reagents for performing transformation ofthe cells and reagents for testing the transgenic cells for geneexpression, to name a few.

The present invention includes transgenic plants comprising one or morecopies of a sucrose metabolizing enzyme-encoding gene, or nucleic acidsequences that inhibit the production or function of a plant'sendogenous sucrose metabolizing enzymes. This is accomplished bytransforming plant cells with a transgene that comprises part of all ofa sucrose metabolizing enzyme coding sequence, or mutant, antisense orvariant thereof, including RNA, controlled by either native orrecombinant regulatory sequences, as described below. Transgenic plantscoffee species are preferred, including, without limitation, C.abeokutae, C. arabica, C. arnoldiana, C. aruwemiensis, C. bengalensis,C. canephora, C. congensis C. dewevrei, C. excelsa, C. eugenioides, andC. heterocalyx, C. kapakata, C. khasiana, C. liberica, C. moloundou, C.rasemosa, C. salvatrix, C. sessfflora, C. stenophylla, C.travencorensis, C. wightiana and C. zanguebariae. Plants of any speciesare also included in the invention; these include, but are not limitedto, tobacco, Arabidopsis and other “laboratory-friendly” species, cerealcrops such as maize, wheat, rice, soybean barley, rye, oats, sorghum,alfalfa, clover and the like, oil-producing plants such as canola,safflower, sunflower, peanut, cacao and the like, vegetable crops suchas tomato tomatillo, potato, pepper, eggplant, sugar beet, carrot,cucumber, lettuce, pea and the like, horticultural plants such as aster,begonia, chrysanthemum, delphinium, petunia, zinnia, lawn andturfgrasses and the like.

Transgenic plants can be generated using standard plant transformationmethods known to those skilled in the art. These include, but are notlimited to, Agrobacterium vectors, polyethylene glycol treatment ofprotoplasts, biolistic DNA delivery, UV laser microbeam, gemini virusvectors or other plant viral vectors, calcium phosphate treatment ofprotoplasts, electroporation of isolated protoplasts, agitation of cellsuspensions in solution with microbeads coated with the transformingDNA, agitation of cell suspension in solution with silicon fibers coatedwith transforming DNA, direct DNA uptake, liposome-mediated DNA uptake,and the like. Such methods have been published in the art. See, e.g.,Methods for Plant Molecular Biology (Weissbach & Weissbach, eds., 1988);Methods in Plant Molecular Biology (Schuler & Zielinski, eds., 1989);Plant Molecular Biology Manual (Gelvin, Schilperoort, Verma, eds.,1993); and Methods in Plant Molecular Biology—A Laboratory Manual(Maliga, Klessig, Cashmore, Gruissem & Varner, eds., 1994).

The method of transformation depends upon the plant to be transformed.Agrobacterium vectors are often used to transform dicot species,Agrobacterium binary vectors include, but are not limited to, BIN19 andderivatives thereof, the pBI vector series, and binary vectors pGA482,pGA492, pLH7000 (GenBank Accession AY234330) and any suitable one of thepCAMBIA vectors (derived from the pPZP vectors constructed byHajdukiewicz, Svab & Maliga, (1994) Plant Mol Biol 25: 989-994,available from CAMBIA, GPO Box 3200, Canberra ACT 2601, Australia or viathe worldwide web at CAMBIA.org). For transformation of monocot species,biolistic bombardment with particles coated with transforming DNA andsilicon fibers coated with transforming DNA are often useful for nucleartransformation. Alternatively, Agrobacterium “superbinary” vectors havebeen used successfully for the transformation of rice, maize and variousother monocot species.

DNA constructs for transforming a selected plant comprise a codingsequence of interest operably linked to appropriate 5′ regulatorysequences (e.g., promoters and translational regulatory sequences) and3′ regulatory sequences (e.g., terminators). In a preferred embodiment,a sucrose metabolizing enzyme-encoding sequence under control of itsnatural 5′ and 3′ regulatory elements is utilized. In other embodiments,sucrose metabolizing enzyme-encoding and regulatory sequences areswapped to alter the sugar profile of the transformed plant for aphenotypic improvement, e.g., in flavor, aroma or other feature.

In an alternative embodiment, the coding region of the gene is placedunder a powerful constitutive promoter, such as the Cauliflower MosaicVirus (CaMV) 35S promoter or the figwort mosaic virus 35S promoter.Other constitutive promoters contemplated for use in the presentinvention include, but are not limited to: T-DNA mannopine synthetase,nopaline synthase and octopine synthase promoters. In other embodiments,a strong monocot promoter is used, for example, the maize ubiquitinpromoter, the rice actin promoter or the rice tubulin promoter (Jeon etal., Plant Physiology. 123: 1005-14, 2000).

Transgenic plants expressing sucrose metabolizing enzyme-codingsequences under an inducible promoter are also contemplated to be withinthe scope of the present invention. Inducible plant promoters includethe tetracycline repressor/operator controlled promoter, the heat shockgene promoters, stress (e.g., wounding)-induced promoters, defenseresponsive gene promoters (e.g. phenylalanine ammonia lyase genes),wound induced gene promoters (e.g. hydroxyproline rich cell wall proteingenes), chemically-inducible gene promoters (e.g., nitrate reductasegenes, glucanase genes, chitinase genes, etc.) and dark-inducible genepromoters (e.g., asparagine synthetase gene) to name a few.

Tissue specific and development-specific promoters are also contemplatedfor use in the present invention. Non-limiting examples of seed-specificpromoters include Cim1 (cytokinin-induced message), cZ19B1 (maize 19 kDazein), milps (myo-inositol-1-phosphate synthase), and celA (cellulosesynthase) (U.S. application Ser. No. 09/377,648), bean beta-phaseolin,napin, beta-conglycinin, soybean lectin, cruciferin, maize 15 kDa zein,22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, andglobulin 1, soybean 11S legumin (Bäumlein et al., 1992), and C.canephora 11S seed storage protein (Marraccini et al., 1999, PlantPhysiol. Biochem. 37: 273-282). See also WO 00/12733, whereseed-preferred promoters from end1 and end2 genes are disclosed. OtherCoffea seed specific promoters may also be utilized, including but notlimited to the oleosin gene promoter described in commonly-owned,co-pending Provisional Application No. 60/696,445 and the dehyrdin genepromoter described in commonly-owned, co-pending Provisional ApplicationNo. 60/696,890. Examples of other tissue-specific promoters include, butare not limited to: the ribulose bisphosphate carboxylase (RuBisCo)small subunit gene promoters (e.g., the coffee small subunit promoter asdescribed by Marraccini et al., 2003) or chlorophyll a/b binding protein(CAB) gene promoters for expression in photosynthetic tissue; and theroot-specific glutamine synthetase gene promoters where expression inroots is desired.

The coding region is also operably linked to an appropriate 3′regulatory sequence. In embodiments where the native 3′ regulatorysequence is not use, the nopaline synthetase polyadenylation region maybe used. Other useful 3′ regulatory regions include, but are not limitedto the octopine synthase polyadenylation region.

The selected coding region, under control of appropriate regulatoryelements, is operably linked to a nuclear drug resistance marker, suchas kanamycin resistance. Other useful selectable marker systems includegenes that confer antibiotic or herbicide resistances (e.g., resistanceto hygromycin, sulfonylurea, phosphinothricin, or glyphosate) or genesconferring selective growth (e.g., phosphomannose isomerase, enablinggrowth of plant cells on mannose). Selectable marker genes include,without limitation, genes encoding antibiotic resistance, such as thoseencoding neomycin phosphotransferase II (NEO), dihydrofolate reductase(DHFR) and hygromycin phosphotransferase (HPT), as well as genes thatconfer resistance to herbicidal compounds, such as glyphosate-resistantEPSPS and/or glyphosate oxidoreducatase (GOX), Bromoxynil nitrilase(BXN) for resistance to bromoxynil, AHAS genes for resistance toimidazolinones, sulfonylurea resistance genes, and2,4-dichlorophenoxyacetate (2,4-D) resistance genes.

In certain embodiments, promoters and other expression regulatorysequences encompassed by the present invention are operably linked toreporter genes. Reporter genes contemplated for use in the inventioninclude, but are not limited to, genes encoding green fluorescentprotein (GFP), red fluorescent protein (DsRed), Cyan Fluorescent Protein(CFP), Yellow Fluorescent Protein (YFP), Cerianthus Orange FluorescentProtein (cOFP), alkaline phosphatase (AP), β-lactamase, chloramphenicolacetyltransferase (CAT), adenosine deaminase (ADA), aminoglycosidephosphotransferase (neo^(r), G418^(r)) dihydrofolate reductase (DHFR),hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ(encoding α-galactosidase), and xanthine guaninephosphoribosyltransferase (XGPRT), Beta-Glucuronidase (gus), PlacentalAlkaline Phosphatase (PLAP), Secreted Embryonic Alkaline Phosphatase(SEAP), or Firefly or Bacterial Luciferase (LUC). As with many of thestandard procedures associated with the practice of the invention,skilled artisans will be aware of additional sequences that can servethe function of a marker or reporter.

Additional sequence modifications are known in the art to enhance geneexpression in a cellular host. These modifications include eliminationof sequences encoding superfluous polyadenylation signals, exon-intronsplice site signals, transposon-like repeats, and other suchwell-characterized sequences that may be deleterious to gene expression.Alternatively, if necessary, the G/C content of the coding sequence maybe adjusted to levels average for a given coffee plant cell host, ascalculated by reference to known genes expressed in a coffee plant cell.Also, when possible, the coding sequence is modified to avoid predictedhairpin secondary mRNA structures. Another alternative to enhance geneexpression is to use 5′ leader sequences. Translation leader sequencesare well known in the art, and include the cis-acting derivative(omega′) of the 5′ leader sequence (omega) of the tobacco mosaic virus,the 5′ leader sequences from brome mosaic virus, alfalfa mosaic virus,and turnip yellow mosaic virus.

Plants are transformed and thereafter screened for one or moreproperties, including the presence of the transgene product, thetransgene-encoding mRNA, or an altered phenotype associated withexpression of the transgene. It should be recognized that the amount ofexpression, as well as the tissue- and temporal-specific pattern ofexpression of the transgenes in transformed plants can vary depending onthe position of their insertion into the nuclear genome. Such positionaleffects are well known in the art. For this reason, several nucleartransformants should be regenerated and tested for expression of thetransgene.

Methods:

The nucleic adds and polypeptides of the present invention can be usedin any one of a number of methods whereby the protein products can beexpressed in coffee plants in order that the proteins may play a role inthe enhancement of the flavor and/or aroma of the coffee beverage orcoffee products ultimately produced from the bean of the coffee plantexpressing the protein.

There is a strong correlation between the sucrose concentration in greenbeans and high quality coffee (Russwurm, 1969; Holscher and Steinhart,1995; Badoud, 2000; Illy and Viani, 1995; Leloup et al., 2003).Improvement of coffee grain sucrose content can be obtained by (1)classical breeding or (2) genetic engineering techniques, and bycombining these two approaches. Both approaches have been considerablyimproved by the isolation and characterization of sucrosemetabolism-related genes in coffee, in accordance with the presentinvention. For example, the sucrose metabolism enzyme-encoding genes maybe genetically mapped and Quantitative Trait Loci (QTL) involved incoffee flavor can be identified. It would be then be possible todetermine if such QTL correlate with the position of sucrose relatedgenes. It is also possible to identify alleles (haplotypes), for genesaffecting sucrose metabolism and examine if the presence of specifichaplotypes are strongly correlated with high sucrose. These “highsucrose” markers can be used to advantage in marker assisted breedingprograms. A third advantage of isolating polynucleotides involved insucrose metabolism is described in detail in the Examples. It is togenerate expression data for the genes during coffee bean maturation invarieties with high and low sucrose levels. This information is used todirect the choice of genes to use in genetic manipulation aimed atgenerating novel transgenic coffee plants that have increased sucroselevels in the mature bean, as described in detail below.

In one aspect, the present invention features methods to alter thesucrose metabolizing enzyme profile, or sugar profile, in a plant,preferably coffee, comprising increasing or decreasing an amount oractivity of one or more sucrose metabolizing enzymes in the plant. Forinstance, in one embodiment of the invention, a sucrose metabolizingenzyme-encoding gene under control of its own expression-controllingsequences is used to transform a plant for the purpose of increasingproduction of that sucrose metabolizing enzyme in the plant.Alternatively, a sucrose metabolizing enzyme-encoding region is operablylinked to heterologous expression controlling regions, such asconstitutive or inducible promoters.

In view of the fact that it has been possible to increase the sucroselevels in the pericarp of tomato by the constitutive over-expression ofSPS, one preferred embodiment of the present invention comprisestransforming coffee plants with an SPS-encoding polynucleotide, such asSEQ ID NO:2, for the purpose of over-producing that coffee SPS invarious tissues of coffee. In one embodiment, coffee plants areengineered for a general increase in SPS activity, e.g., through the useof a promoter such as the RuBisCo small subunit (SSU) promoter or theCaMV35S promoter. In another embodiment designed to limit the effects ofover-expressing SPS only to the sink organ of interest, i.e., the grain,a grain-specific promoter may be utilized, particularly one of theCoffea grain-specific promoters described above.

The sucrose profile of a plant may be enhanced by modulating theproduction, or activity, of one or more sucrose metabolizing enzymes inthe plant, such as coffee. Additionally, plants expressing enhancesucrose levels may be screened for naturally-occurring variants of thesucrose metabolizing enzymes. For instance, loss-of-function (null)mutant plants may be created or selected from populations of plantmutants currently available. It will also be appreciated by those ofskill in the art that mutant plant populations may also be screened formutants that over-express a particular sucrose metabolizing enzyme,utilizing one or more of the methods described herein. Mutantpopulations can be made by chemical mutagenesis, radiation mutagenesis,and transposon or T-DNA insertions, or targeting induced local lesionsin genomes (TILLING, see, e.g., Henikoff et al., 2004, Plant Physiol,135(2): 630-636; Gilchrist & Haughn, 2005, Curr. Opin. Plant Biol. 8(2):211-215). The methods to make mutant populations are well known in theart.

Of particular interest are mutants of sucrose metabolizing enzymes thathave select mutations that alter the post-translational modification ofthe enzyme, which may affect the enzymatic activity or substratespecificity of the enzyme. Post-translational modification is understoodin the art to include a number of modifications to a protein that occursin eukaryotic cells after translation of the protein and can include,among others, glycosylation, alkylation, and phosphorylation of theprotein. In some examples, the sucrose metabolizing enzyme SPS, whichhas been found to have potential phosphorylation sites at Ser150, Ser221and Ser415, may have phosphorylation sites removed by the introductionof point-mutations at any one or a combination of the potentialphosphorylation sites. Through these point-mutations, thephosphorylation pattern of the enzyme can be modified, thus modifyingthe activity of the enzyme. Ser150 is thought to be a regulation sitefor the enzyme SPS; thus, by removing this phosphorylation site bysite-directed mutagenesis, the activity of SPS may be enhanced.Additionally, Ser415 is thought to have an antagonizing relationship tothe regulatory effect of phosphorylation of Ser150; therefore, removalof this phosphorylation site (Ser415) could further regulate theactivity of SPS. Further, phosphorylation at Ser221 of SPS is thought toalso inhibit the activity of SPS. Removal of this phosphorylation site(Ser221) could also enhance the activity of SPS.

The nucleic acids of the invention can be used to identify mutant formsof sucrose metabolizing enzymes in various plant species. In speciessuch as maize or Arabidopsis, where transposon insertion lines areavailable, oligonucleotide primers can be designed to screen lines forinsertions in the sucrose metabolizing enzyme genes. Through breeding, aplant line may then be developed that is heterozygous or homozygous forthe interrupted gene.

A plant also may be engineered to display a phenotype similar to thatseen in null mutants created by mutagenic techniques. A transgenic nullmutant can be created by a expressing a mutant form of a selectedsucrose metabolizing enzyme protein to create a “dominant negativeeffect.” While not limiting the invention to any one mechanism, thismutant protein will compete with wild-type protein for interactingproteins or other cellular factors. Examples of this type of “dominantnegative” effect are well known for both insect and vertebrate systems(Radke et al, 1997, Genetics 145: 163-171; Kolch et al., 1991, Nature349: 426-428).

Another kind of transgenic null mutant can be created by inhibiting thetranslation of sucrose metabolizing enzyme-encoding mRNA by“post-transcriptional gene silencing.” The sucrose metabolizingenzyme-encoding gene from the species targeted for down-regulation, or afragment thereof, may be utilized to control the production of theencoded protein. Full-length antisense molecules can be used for thispurpose. Alternatively, antisense oligonucleotides targeted to specificregions of the mRNA that are critical for translation may be utilized.The use of antisense molecules to decrease expression levels of apre-determined gene is known in the art. Antisense molecules may beprovided in situ by transforming plant cells with a DNA construct which,upon transcription, produces the antisense RNA sequences. Suchconstructs can be designed to produce full-length or partial antisensesequences. This gene silencing effect can be enhanced by transgenicallyover-producing both sense and antisense RNA of the gene coding sequenceso that a high amount of dsRNA is produced (for example see Waterhouseet al., 1998, PNAS 95: 13959-13964). In this regard, dsRNA containingsequences that correspond to part or all of at least one intron havebeen found particularly effective. In one embodiment, part or all of thesucrose metabolizing enzyme-encoding sequence antisense strand isexpressed by a transgene. In another embodiment, hybridizing sense andantisense strands of part or all of the sucrose metabolizingenzyme-encoding sequence are transgenically expressed.

In another embodiment, sucrose metabolizing enzyme-encoding genes may besilenced through the use of a variety of other post-transcriptional genesilencing (RNA silencing) techniques that are currently available forplant systems. RNA silencing involves the processing of double-strandedRNA (dsRNA) into small 21-28 nucleotide fragments by an RNase H-basedenzyme (“Dicer” or “Dicer-like”). The cleavage products, which are siRNA(small interfering RNA) or miRNA (micro-RNA) are incorporated intoprotein effector complexes that regulate gene expression in asequence-specific manner (for reviews of RNA silencing in plants, seeHoriguchi, 2004, Differentiation 72: 65-73; Baulcombe, 2004, Nature 431:356-363; Herr, 2004, Biochem. Soc. Trans. 32: 946-951).

Small interfering RNAs may be chemically synthesized or transcribed andamplified in vitro, and then delivered to the cells. Delivery may bethrough microinjection (Tuschl T et al., 2002), chemical transfection(Agrawal N et al., 2003), electroporation or cationic liposome-mediatedtransfection (Brummelkamp T R et al., 2002; Elbashir S M et al., 2002),or any other means available in the art, which will be appreciated bythe skilled artisan. Alternatively, the siRNA may be expressedintracellularly by inserting DNA templates for siRNA into the cells ofinterest, for example, by means of a plasmid, (Tuschl T et al., 2002),and may be specifically targeted to select cells. Small interfering RNAshave been successfully introduced into plants, (Klahre U et al., 2002).

A preferred method of RNA silencing in the present invention is the useof short hairpin RNAs (shRNA). A vector containing a DNA sequenceencoding for a particular desired siRNA sequence is delivered into atarget cell by any common means. Once in the cell, the DNA sequence iscontinuously transcribed into RNA molecules that loop back on themselvesand form hairpin structures through intramolecular base pairing. Thesehairpin structures, once processed by the cell, are equivalent to siRNAmolecules and are used by the cell to mediate RNA silencing of thedesired protein. Various constructs of particular utility for RNAsilencing in plants are described by Horiguchi, 2004, supra. Typically,such a construct comprises a promoter, a sequence of the target gene tobe silenced in the “sense” orientation, a spacer, the antisense of thetarget gene sequence, and a terminator.

Yet another type of synthetic null mutant can also be created by thetechnique of “co-suppression” (Vaucheret et al., 1998, Plant J. 16(6):651-659). Plant cells are transformed with a copy of the endogenous genetargeted for repression. In many cases, this results in the completerepression of the native gene as well as the transgene. In oneembodiment, a sucrose metabolizing enzyme-encoding gene from the plantspecies of interest is isolated and used to transform cells of that samespecies.

Mutant or transgenic plants produced by any of the foregoing methods arealso featured in accordance with the present invention. Preferably, theplants are fertile, thereby being useful for breeding purposes. Thus,mutant or plants that exhibit one or more of the aforementioneddesirable phenotypes can be used for plant breeding, or directly inagricultural or horticultural applications. They will also be of utilityas research tools for the further elucidation of the participation ofsucrose metabolizing enzymes and its affects on sucrose levels, therebyaffecting the flavor, aroma and other features of coffee seeds. Plantscontaining one transgene or a specified mutation may also be crossedwith plants containing a complementary transgene or genotype in order toproduce plants with enhanced or combined phenotypes.

The following examples are provided to describe the invention in greaterdetail. The examples are for illustrative purposes, and are not intendedto limit the invention.

Example 1 Materials and Methods for Subsequent Examples

Plant Material. Tissues from either leaves, flowers, stem, roots, orcherries were harvested at different stages of development from Coffeaarabica L. cv, Caturra T-2308 grown under greenhouse conditions (25° C.,70% RH) in Tours, France, and from Coffea canephora (robusta) BP-409grown in the field at the Indonesian Coffee and Cacao Research Center(ICCRI), Indonesia. FRT05 (Robusta) and CCCA12 (Arabica) were obtainedfrom trees cultivated in Centre Quito, Ecuador. The fruit was harvestedat defined stages and frozen immediately in liquid nitrogen, and thenpackaged in dry ice for transport. Tissues were stored at −80° C. untiluse.

Genomic DNA preparation. Leaves from BP-409 were harvested fromgreenhouse-grown trees at Tours, France. Tissue was frozen immediatelyin liquid nitrogen and reduced in fine powder. Genomic DNA was preparedaccording to Crouzillat et al., 1996.

PCR amplification of partial coffee SPS Gene. Degeneratedoligonucleotides SPS-3 (5′ ggNcgNgaYtetgaYacNggtgg3′) (SEQ ID NO.:20)and SPS-4 (5′ tggacgacaYtcNccaaaNgcYttNac3′) (SEQ ID NO.:21) were madefrom the conserved sequence of sucrose-phosphate synthase deduced fromthe alignment set forth in FIG. 4 and used as primers in PCRamplification. PCR reactions were performed in a 50 μl reaction volumewith 100 ng genomic DNA, 0.5 μM of each primer, 200 μM of dNTPs, 1× Taqpolymerase buffer and 1 U of TaqDNA polymerase (TAKARA). After apre-denaturing step at 94° C. for 5 min, the amplification consisted of30 cycles of 1 min at 94° C., 1 min at 12 different temperatures (from45° C. to 56° C.) and 2 min at 72° C. The resulting PCR fragments wereseparated and purified by agarose gel electrophoresis. PCR fragment fromthe major bands was purified, cloned and sequenced.

Isolation of CcSPS1 and CcSPS2 partial cDNA sequences. In order toverify if CcSPS1 and CcSPS2 genes were expressed, specific primers weredesigned based on potential coding sequences identified on the partialgenomic CcSPS1 and CeSPS2 sequences (SEQ ID NOS: 4 and 5, respectively).Two sets of primers, cDNAC1-1 (^(5′)AACTTGCAAGGGCTTTAGGT^(3′)) (SEQ IDNO.:22), cDNAC1-2 (^(5′)AAGGGCTAGTATCATAGGCT^(3′)) (SEQ ID NO.:23) andcDNAD1-1 (^(5′)AGCTTGCTAAGGCACTTGCT^(3′)) (SEQ ID NO.:24), cDNAD1-2(^(5′)CAATGCTAGAATCATTGGCT^(3′)) (SEQ ID NO.:25) were used to amplifypartial CcSPS1 and CcSPS2 cDNA sequences respectively by PCR usingvarious cDNA samples prepared as described below.

Universal Genome Walker. Genomic DNA from BP409 was hydrolyzed with fourdifferent restriction enzymes (Drat, EcoRV, PvuI, StuI) and theresulting fragments were ligated blunt-end to the GenomeWalker Adaptorprovided by the Universal GenomeWalker kit (BD Biosciences). Bothreactions were carried out in accordance with the kit user manual. Thefour libraries were then employed as templates in PCR reactions usingSPS-GSP (gene-specific primers) (Table 1) The reaction mixturescontained 1 μl of GenomeWalker library template, 10 nmol of each dNTP,50 pmol of each primer and 2.5 units of DNA polymerase in a final volumeof 50 μl with the appropriate buffer. The following conditions were usedfor the first PCR: after pre-denaturing at 95° C. for 2 min, the firstseven cycles were performed at a denaturing temperature of 95° C. for 30s, followed by an annealing and elongation step at 72° C. for 3 min. Afurther 35 cycles were carried out, changing the annealing/elongationtemperature to 67° C. for 3 min. Products from the first amplificationusing the primer pair AP1/C1-GW (Genome Walker) served as template forthe second PCR using AP2/C1-GWN (Genome Walker Nested primer), with AP2and C1-GWN as primers. The second PCR used 2 μl of the firstamplification reaction (undiluted and different dilutions up to 1:50),and was performed as described above for the first reaction, with theexception that the second reaction used only 25 cycles of amplification.The resulting PCR fragments were separated and purified by agarose gelelectrophoresis. PCR fragment from the major bands was purified, clonedand sequenced.

TABLE 1 List of primers used for Genome Walker experiments SequencePrimers Sequences Identifier AP1 ^(5′) gtaatacgactcactataggSEQ ID NO.: 26 gc ^(3′) AP2 ^(5′) actatagggcacgcgtggt ^(3′)SEQ ID NO.: 27 C1-GW1 ^(5′) tacttccagtgatgatacctg SEQ ID NO.: 28cctcgta ^(3′) C1-GWN1 ^(5′) tctaggaggcagcatctcagt SEQ ID NO.: 29gggttca ^(3′) C1-GW3 ^(5′) ccggatccacatatttgggga SEQ ID NO.: 30gaggtct ^(3′) C1-GWN3 ^(5′) tggtgtcatgcagataatgcg SEQ ID NO.: 31ctacttc ^(3′) C1-GW6 ^(5′) gcaatcgacccctattgctct SEQ ID NO.: 32caccatgt ^(3′) C1-GWN6 ^(5′) agtcttcagacatatcagcaa SEQ ID NO.: 33ctgcttc ^(3′) C1-GW7 ^(5′) gtgagctctctgtggttgatg SEQ ID NO.: 34ttgttga ^(3′) C1-GWN7 ^(5′) gtttcgaattctggctcaatg SEQ ID NO.: 35caaccact ^(3′)

DNA sequence analysis. For DNA sequencing, recombinant plasmid DNA wasprepared and sequenced according to standard methods. Computer analysiswas performed using DNA Star (Lasergene) software. Sequence homologieswere verified against GenBank databases using BLAST programs (Altschulet al. 1990).

cDNA preparation, RNA was extracted from different tissues i.e. root,stem, leaves, flowers, pericarp and grain at four different maturationstages SG (small green), LG (large green), Y (yellow), R (red), asdescribed previously (Benamor and Mc Carthy, 2003). cDNA was preparedfrom total RNA and oligo dT(18) (Sigma) as follows: 1 μg total RNAsample plus 50 ng oligo dT was made up to 12 μl final volume withDEPC-treated water. This mixture was subsequently incubated at 70° C.for 10 min and then rapidly cooled on ice. Next, 4 μl of first strandbuffer (5×, Invitrogen), 2 μl of DTT (0.1 M, Invitrogen) and 1 μl ofdNTP mix (10 mM each, Invitrogen) were added. These reaction mixes werepreincubated at 42° C. for 2 min before adding 1 μl-SuperScript IIIRnase H-Reverse transcriptase (200 U/μl, Invitrogen). Subsequently, thetubes were incubated at 42° C. for 50 min, followed by enzymeinactivation by heating at 70° C. for 10 min. The cDNA samples generatedwere then diluted one hundred fold and 5 μl of the diluted cDNA wereused for Q-PCR.

Full length SPS cDNA amplification. In order to amplify full lengthCcSPS1 cDNA, two primers:

-   cDNAC1-am3 (^(5′)ATGGCGGGAAATGACTGGATAAACAGTTAC^(3′)) (SEQ ID    NO.:36) and-   cDNAC1-am4 (^(5′)CTAGCTTTTGAGAACCCCTAGCTTTTCCAAC^(3′)) (SEQ ID    NO.:37)    have been designed based on the CcSPS1 genomic sequence. These two    primers have been used to perform PCR reaction using methods as    described above. The single fragment obtained has been purified from    agarose gel, cloned and sequenced.

Quantitative-PCR. TaqMan-PCR was carried out as recommended by themanufacturer (Applied Biosystems, Perkin-Elmer). All reactions contained1× TaqMan buffer (Perkin-Elmer) and 5 mM MgCl2, 200 μM each of dATP,dCTP, dGTP and dTTP, and 0.625 units of AmpliTaq Gold polymerase, PCRwas carried out using 800 nM of each gene specific primers, forward andreverse, and 200 nM TaqMan probe. Primers and probes were designed usingPRIMER EXPRESS software (Applied Biosystems, Table 2). Reaction mixtureswere incubated for 2 min at 50° C., 10 min at 95° C., followed by 40amplification cycles of 15 sec at 95° C./1 min at 60° C. Samples werequantified in the GeneAmp 7500 Sequence Detection System (AppliedBiosystems). Transcript levels were determined using rp139 as a basis ofcomparison.

TABLE 2 List of primers and probes used for Q-PCR Primers and SequenceProbes Sequences Identifier rp139-F1 ^(5′) GAACAGGCCCATCCCTTATTSEQ ID NO.: 38 G ^(3′) rp139-R1 ^(5′) CGGCGCTTGGCATTGTA ^(3′)SEQ ID NO.: 39 rp139-MGB1 ^(5′) ATGCGCACTGACAACA ^(3′) SEQ ID NO.: 40CcSPS1-R1 ^(5′) CGCAATGTTAGCTGTTATG ^(3′) SEQ ID NO.: 41 CcSPS1-F1^(5′) GAAATTGCGGGCTAGGATCA ^(3′) SEQ ID NO.: 42 CcSPS1-^(5′) GCCATTCGAGGCATGAATC SEQ ID NO.: 43 MGB1 T ^(3′) CcSS2-F1^(5′) TTCTGCCAGTCTTGCCTTTCT SEQ ID NO.: 44 T ^(3′) CcSS2-R1^(5′) CCTAATTGACACTTGAACAGG SEQ ID NO.: 45 GACTA ^(3′) CcSS2-MGB1^(5′) TTGTTGGTTGGTTGTGTCT ^(3′) SEQ ID NO.: 46

Soluble Sugars quantification. Grain tissues were separated frompericarp and hulls. The grain were homogenized in cryogenic grinder withliquid nitrogen and the powder obtained was lyophilized for 48 hours(Lyolab bII, Secfroid). Each sample was weighed and suspended in 70 mlof double-distilled water previously pre-heated to 70° C., then shakenvigorously and incubated for 30 min at 70° C. After cooling to roomtemperature, the sample was brought to 100 ml by addingdoubled-distilled water, and then paper filtered (Schleicher and Schuellfilter paper 597.5), Sugars of extracted coffee grain tissues wereseparated by HPAE-PED according to Locher et al., 1998 using a Dionex PA100 (4×250 mm) column. Sugar concentration was expressed in g per 100 gof DW (dry weight).

Enzymatic Activity analysis. Sucrose synthase activity was measuredaccording to Lafta and Lorenzen (1995). Sucrose phosphate synthaseactivity was measured according to Trevanion et al. (2004).

Example 2 Identification of cDNA Encoding Enzymes of Sucrose Metabolism

More than 47,000 EST sequences were identified from several coffeelibraries made with RNA isolated from young leaves and from the grainand pericarp tissues of cherries harvested at different stages ofdevelopment. Overlapping ESTs were subsequently “clustered” into“unigenes” (ie contigs) and the unigene sequences were annotated bydoing a BLAST search of each individual sequence against the NCBInon-redundant protein database.

Enzymes directly involved in the synthesis and degradation of sucrosehave been widely studied in plants, and especially during fruit, tuber,and seed development in plants such as tomato (Lycopersicon esculentum),potato (Solanum tuberosum) and corn (Zea mays). DNA sequences coding forall known key proteins involved in sucrose synthesis and degradationhave been identified and characterized in several species and areavailable in GenBank. Accordingly, the known sequences of plant enzymes,especially sequences from organisms closely related to coffee (e.g.,tomato and potato), were used to find similar sequences present in theabove-described EST libraries and in other coffee cDNA libraries. Tosearch the aforementioned EST collection, protein sequences of tomatoand potato were used in a tBLASTn search of the “unigene” set 3 asdescribed in Example 1. Those in-silica “unigenes” whose open readingframes showed the highest degree of identity with the “query” sequencewere selected for further study. In some cases, the selected “unigenes”contained at least one EST sequence that potentially represented a fulllength cDNA clone, and that clone was then selected for re-sequencing toconfirm both its identity and the “unigene” sequence.

A. Sucrose Synthase CcSS2 (SEQ ID NO:1)

The clone A5-1540, which is highly related to sucrose synthase 2 (SS2)from tomato (Lycopersicon esculentum, NCBI Protein Identifier No.CAA09681), was found in a coffee cDNA collection (as opposed to the ESTcollection). The protein encoded by A5-1540 clone is 88.6% identical toSS2 from tomato and is apparently full length (FIG. 2). The cDNA insertis 3048 bp long, and is characterized by a 2427 bp ORF which starts atposition 248 and finishes position 2668. This sequence is referred asSEQ ID NO 1. The deduced protein (SEQ ID NO:8) is 805 aa long, with apredicted molecular weight of 92.6 kDa. The protein sequence encoded bythe clone A5-1540 was analyzed for similarity to all publicly availableprotein sequences contained in the NCBI nonredundant database. Theresulting alignment of the most closely related sequences is presentedin FIG. 2. As can be seen from the figure, residues 7-554 of SEQ ID NO:8comprises a domain that characterizes members of the sucrose synthasefamily, and residues 565-727 is a domain that characterizes glycosyltransferase group 1.

As well as being closely related to SS2 of tomato, it is also closelyrelated to potato SS2 (89% identity; NCBI Protein IdentifierNo.AA034668). Subsequently, unigene #97089 was found in the ESTdatabase, and that sequence was determined to correspond to the samesequence as A5-1540. However, the longest clone in this unigene is over1,400 nucleotides shorter than the clone A5-1540, and thus the ESTdatabase does not appear to contain a full length clone. Inmonocotyledons (maize and sorghum), SS is encoded by threedifferentially expressed nonallelic loci, sus1, sus2 and sus3 (Choureyet al., 1991, Huang et al., 1996, Carlson et al. 2002). Mostdicotyledonous species contain two nonallelic SS genes, which arefunctional analogs of two classes of SS genes from monocotyledons (Fu etal., 1995). Homology results show that the coffee sequence is closest tothe SS2 sequence of potato and the protein encoded by clone A5-1540therefore was designated CcSS2, for Coffea canephora sucrose synthase 2,herein.

B. Sucrose Phosphate Synthase CcSPS1 (SEQ ID NO:2)

The protein sequence of sucrose phosphate synthase (SPSLE1, NCBI ProteinIdentifier No. AAC24872) from tomato (Lycopersicon esculentum) was usedto perform a similarity search of the EST-based unigene set using thetBLASTn algorithm. No unigene was found that could potentially code foran SPS protein.

Due to lack of a match in the available database, a partial sequencefrom the Coffea canephora BP-409 genome was amplified using degenerateoligonucleotides. By alignment of different SPS protein sequences, itwas possible to identify a highly conserved domain and to designdegenerate primers corresponding to the protein sequence encoded at theend of exon 4 and at the beginning of exon 7 (Fragment C1 in FIG. 3).

Two different PCR fragments of 1500 and 2000 bp, respectively, wereamplified using PCR and degenerate oligonucleotides. After sequencing ofboth genomic sequences, an alignment of putative encoded proteinsequences with the tomato protein sequence SPSLE1 showed isolation ofpartial sequences from two different coffee SPS genes, CcSPS1 andCcSPS2. The fragments corresponding to CcSPS1 and CcSPS2 partialsequence were 1937 and 1564 bp long, respectively. The protein sequencesencoded by partial clones of CcSPS1 and CcSPS2 were found to share ahigh degree of homology. Introns 4, 5 and 6 were shorter for CcSPS2,thus explaining the difference in size between the two amplifiedfragments (data not shown). Preliminary expression analysis indicatedthat CcSPS2 was not expressed, while CcSPS1 was expressed in varioustissues, including grain. Therefore, the CcSPS1 gene was examinedfurther.

Using several rounds of primer directed genome walking using the GenomeWalker™ technique, a full length genomic sequence for the CcSPS1 genewas amplified. A schematic representation of the CcSPS1 gene is shown inFIG. 3. The gene is characterized by 13 exons and 12 introns. The CcSPS1gene is 7581 bp long (from initiation codon ATG to stop codon TAG) isreferred to as SEQ ID NO:6. Using specific primers deduced from CcSPS1genomic sequence, the CcSPS1 full length cDNA was amplified by RT-PCR.Several RNA samples were used, positive amplification corresponding tothe full length cDNA sequence was only obtained using RNA extracted frompericarp at yellow stage from robusta. The CcSPS1 cDNA is 3150 bp longand this DNA sequence is referred as SEQ ID NO: 2. The deduced protein,SEQ ID NO: 9, is 1049 aa long, with a predicted molecular weight of117.9 kDa. The protein sequence encoded by the CcSPS1 cDNA shows a veryhigh level of homology (82.6%) with the tomato SPSLE1 protein sequence(FIG. 4). In addition, residues 168-439 of SEQ ID NO:9 characterizemembers of the sucrose phosphate synthase family, and residues 467-644characterize glycosyl transferases group 1 family members.

C. Sucrose Phosphate Phosphatase CcSP1 (SEQ ID NO:3)

The protein sequence of sucrose phosphatase (SP, NCBI Protein IdentifierNo. AA033160) from tomato (Lycopersicon esculentum) was used to performa similarity search of the EST-based unigene set using the tBLASTnalgorithm. The ORF of unigene #102159 showed a high degree of homologyto the tomato SP sequence and the single EST (cDNA) in this unigene,clone ccc119n15, was isolated and its insert was fully sequenced. ThecDNA insert of ccc119n15 is apparently full length and was found to be1721 bp long. This sequence is referred as SEQ ID NO 3. The complete ORFsequence of this clone was 1248 bp long, starting at position 135 andfinishing at position 1409. The deduced protein (SEQ ID NO:10) was 415aa long with a predicted molecular weight of 46.7 kDa. Residues 1-408 ofSEQ ID NO:10 characterize members of the sucrose-6F-phosphatephosphohydrolase family. The ORF of ccc119n15 is 81% identical to thetomato SP protein. The protein encoded by ccc119n15 was also analyzedfor similarity to all publicly available protein sequences contained inthe NCBI nonredundant database. The alignment of sequences showing thehighest homologies is presented in FIG. 5. Only one distinct unigene hasbeen found in the coffee cDNA libraries. Several species (includingmaize, tomato, wheat and barley) are known to contain at least two SPgenes; Arabidopsis has four and rice three (Lunn et MacRae, 2003). Basedon homology results presented here, the cDNA clone ccc119n15 clone hasbeen renamed CcSP1 for C. canephora sucrose phosphatase 1.

Example 3 Control of SPS Activity by Reversible Protein Phosphorylation

A major regulatory site of spinach SPS has been identified as Ser 158(McMichael et al., 1993). Phosphorylation of Ser158 is both necessaryand sufficient for the inactivation of SPS in vitro. Similar resultswere shown for the phosphorylation of Ser162 of maize SPS in studies ofmaize leaves, as well as transgenic tobacco expressing the maize SPSgene (Huber et al., 1995). Although the regulatory phosphorylationsequence of spinach SPS is not conserved exactly, all sequencesavailable to date were determined to contain a homologous seryl residue.

The CcSPS1 deduced protein sequence was aligned with other SPS proteinsequences from different species. The serine residue of CcSPS1 mostlikely to be homologous to Ser 158 in spinach was identified as Ser 150(FIG. 4). Most of the residues surrounding the putative phosphorylationsite are consistently conserved among the five aligned proteins, e.g.,there are basic residues at P-3 (R), P-6 (R) and P-8 (R or K) (numberingrelative to Ser, which is position 0). Several, and possibly all, ofthese conserved residues may be important for recognition by a proteinkinase (Huber and Huber, 1996; McMichael et al., 1993). The enzymaticactivity of CcSPS1 could be modulated byphosphorylation/dephosphorylation of Ser150. In one example, the Ser150could be replaced with another conservative replacement amino acid bysite-directed mutagenesis; thereby, eliminating this phosphorylationsite and enhancing SPS activity by eliminating regulation viaphosphorylation.

Recent evidence suggests that there may be a second regulatoryphosphorylation site at Ser 424 of spinach SPS, which is phosphorylatedwhen leaf tissue is subjected to osmotic stress (Toroser and Huber,1997). Phosphorylation of Ser 424 in spinach activates the enzyme,perhaps by antagonizing the inhibitory effect of Ser158 phosphorylation.This site was also determined to be widely conserved among species. Thehomologous site in CcSPS1 protein was determined to be Ser 415. Thesucrose synthesis activity of SPS could be enhanced by placing coffee ina simulated high osmotic stress environment to facilitate thephosphorylation of Ser415.

A third potential phosphorylation site may also exist, inasmuch asrecent results have demonstrated that 14-3-3 proteins can associate withspinach leaf SPS in the presence of Mg²⁺. The effect of this specific14-3-3 protein/SPS interaction was to partially inhibit the SPSactivity. It has been proposed that the 14-3-3 protein may function as ascaffold protein to facilitate the interaction of SPS with otherproteins. The suggested site of interaction in spinach SPS is Ser 229.The homologous region in CcSPS1 is Ser 221 and, notably, this region ofthe protein is strongly conserved (FIG. 4). In one example, the Ser221could be replaced with another conservative replacement amino acid bysite-directed mutagenesis; thereby, eliminating this phosphorylationsite and enhancing SPS activity by eliminating regulation viaphosphorylation.

In summary, SPS enzymatic activity can be regulated by reversibleprotein phosphorylation, and three sites have been shown to be involvedin enzyme activity regulation of spinach or maize enzyme. By alignmentof CcSPS1 with SPS from other species, these putative seryl residueshave been localized in CcSPS1 to Ser 150, Ser 221 and Ser 415.

Example 4 Sugar Accumulation and Enzymatic Activity During Coffee SeedDevelopment

Sugar Quantification. Sugar levels during coffee grain maturation wereexamined in C. canephora variety FRT05 (robusta) and C. Arabica varietyCCCA12 (arabica). These two genotypes were chosen because they have beenfound to possess significantly different levels of sucrose. The amountsof sucrose, glucose and fructose in the FRT05 and CCCA12 coffee grainduring maturation were measured in samples harvested in parallel. Thesame samples were also used for the assays of SPS and SuSy activitydescribed below. The results are shown in FIG. 6.

At the earliest stage of maturity (stage SG), the main free sugar wasfound to be glucose, but the concentration is 10 times higher in arabica(14%) than robusta (1.5%). At the same stage, fructose concentration wasalso higher in arabica (1.5%) than robusta (0.3%), but at a much lowerlevel than glucose. By the end of grain development, concentrations ofglucose and fructose were found to have decreased to very low levels forboth species, with only trace levels being detected at the mature redstage (R). The decrease in fructose and glucose was accompanied by anincrease in sucrose, which approached 100% of total free sugars inmature grains, with higher levels found in arabica (9.82%) than robusta(6.71%). These results represent only free sugar accumulation and do notinclude their modified form, e.g., UDP-G, F6-P and S6-P, which are alsoknown to play a role in sucrose metabolism (FIG. 1).

SuSy and SPS Enzyme Activity. In parallel to the sugar quantification,sucrose synthase (SuSy) and sucrose phosphate synthase (SPS) enzymeactivities were studied in order to determine if there might be a strongcorrelation between free sugar accumulation and these particular enzymeactivities and to elucidate the reason CCCA12 (Arabica) accumulates 30%more sucrose than FRT05 (robusta).

The enzymatic activities of SuSy and SPS were determined similarly foreach of the same development stages. SuSy (EC 2.4.1.13) catalyzes thereversible cleavage of sucrose in the presence of UDP to formUDP-glucose and fructose, while SPS (EC 2.3.1.14) catalyzes thesynthesis of sucrose phosphate and UDP starting from fructose6-phosphate and UDP-Glucose (FIG. 1).

Low SuSy activity was observed in early stage of development (stage SG),with the activity being almost two times higher in arabica (0.007 U)than robusta (0.004) (FIG. 6). SuSy activity rose drastically between SGand LG stage and reached a peak of 0.069 U for arabica and 0.12 U forrobusta. Again, SuSy activity was twice as high for robusta as comparedwith arabica at the LG stage. In the later stage of development, theSuSy activity declined dramatically for both species to reachapproximately similar low levels of activity at the Y stage. Between Yand R stages, SuSy activity remained constant but weak for arabica aswell as for robusta.

Overall, the SuSy activity was clearly higher at all stages in robustathan in arabica. The profiles of SuSy activity during both arabica androbusta grain development were similar to those seen in various otherplants, such as tomato and maize. For those species, it has been shownthat SuSy activity is highly correlated with sucrose unloading capacityfrom the phloem (phenomenon also called sink strength; Sun, et al.,1992; Zrenner et al., 1995). If this correlation exists in coffee grain,this implies that the sink strength of robusta should be higher at thelarge green stage of robusta versus the same stage of arabica.Interestingly, although the peak of SuSy activity reached its highestpoint between SG and LG stage in both species, the sucroseconcentrations of both were not drastically increased. This suggestseither that sucrose is not re-synthesised immediately after import, orthat sucrose is being rapidly funneled into another pathway.

The activity pattern observed for SPS activity during coffee seedmaturation was found to be completely different for arabica and robusta(FIG. 6). At the earliest stage (SG), SPS activity in robusta (0.02 U)was 2.5-fold higher than that observed for arabica (0.008 U). Inrobusta, SPS activity was seen to decrease to undetectable levels at theY stage then rise again to the levels seen in the SG stage at R stage.In contrast, for arabica, SPS activity rose sharply between SG and LGstages, reaching an activity of 0.04 U, and then continued to riseslowly during the final maturation process, reaching 0.052 U at stage R.The fluctuation of SPS activity appeared to be quite high for robusta,while, in contrast, the SPS activity rose gradually during grainmaturation to reach a relatively high level at the R stage in arabica.The difference in the SPS activity levels during grain maturation islikely to be an important contributing factor that leads to sucroseaccumulation that is 30% higher in arabica than robusta.

Example 5 CcSS2, CcSPS1 and CcSP1 mRNA Expression at Different Stages ofCoffee Grain Maturation

To determine if any correlation existed between the enzymatic activityfluctuations seen for SuSy and SPS and the expression of these genesduring coffee bean maturation, the expression of the three genes CcSS2,CcSPS1 and CcSP1 during T2308 (C. arabica, arabica) and BP-409 (C.canephora, robusta) grain development was characterized. For comparativepurposes, the expression of these genes in different coffee tissues,such as leaf, flower and root, was also examined. It is noted that geneexpression analysis was not carried out on the same genotypes as thoseused for enzymatic activities, but comparisons were still made betweenarabica and robusta.

RNA was extracted from BP-409 and T2308 coffee cherries at fourdifferent maturation stages characterized by size and color, i.e. SG(small green), LG (large green), Y (yellow) and R (red or mature). Foreach stage, pericarp and grain were separated before total RNA wasextracted as described in Example 1. Total RNA was also extracted fromother tissues (leaf, root and flower). Gene expression was analyzed byperforming real time RT-PCR (TaqMan, Applied Biosystems). Relativetranscript levels were quantified against an endogenous constitutivetranscript rpl39. The gene specific primers and the TaqMan probes usedare listed in Example 1.

The CcSS2 transcript was highly expressed in robusta grain at theearliest stage of development (6 U of RQ). The level of CcSS2 mRNA thendecreased gradually during coffee grain maturation until beingequivalent to a value of 0.3 U of RQ at the mature stage (R). Arelatively similar expression pattern was seen for arabica grain,although the absolute levels were different. The relative amount ofexpression for CcSS2 was higher in robusta than arabica grain at the SGstage, equivalent at LG stage for both species, and lower in robustathan arabica at the Y stage. Transcript accumulation was slightlygreater in arabica than robusta at the mature stage (R). Except in thesmall green stage, low levels of CcSS2 accumulate in the pericarp ofrobusta and arabica cherries. Similar results (Wang et al, 1994; Carlsonet al., 2002) were obtained previously in tomato and maize, in bothcases there was an early accumulation of SuSy mRNA, followed bydecreasing levels of transcripts being detected as fruit and grainmaturation progressed. Significant levels of CcSS2 transcripts were alsodetected in other coffee tissues such as root, flower and leaf for bothspecies.

The CcSPS1 transcript is expressed at very low levels compared to CcSS2mRNA, with the highest RQ observed being 0.07 U in arabica (FIG. 6).CcSPS1 transcripts were almost undetectable during all stages of robustagrain maturation examined, and the highest expression detected inrobusta was in yellow pericarp and flower (0.02 U). The level of CcSPS1transcripts in the robusta root and leaf tissues were below detection.Interestingly, CcSPS1 transcripts were detected in all the tissuesexamined for arabica, especially in the flower, grain and pericarp. Inarabica, the transcript level increased 10-fold between SG (0.005) and Ystage (0.05 U). In mature grain stage, the level was slightly lower(0.04 U). Overall, the results obtained indicate that CcSPS1 mRNAexpression is significantly higher in arabica than robusta. This mainresult correlates well with the differences in SPS activity, presentedearlier, with the detectable SPS activity being significantly higher inarabica than in robusta.

CcSP1 transcript accumulation was very low in grain and pericarp at allthe maturation stages examined for both species, although expression wasslightly higher in arabica than robusta (FIG. 6).

Generally, arabica grains accumulate more sucrose than do robustagrains. To summarize the results set forth above, chemical analysisshowed that CCCA12 (Arabica) accumulated 30% more sucrose in the maturegrain than FRT05 (robusta). The activity of SuSy and SPS was alsodetermined for both species during coffee bean maturation. Notably, SuSyactivity was found to be higher in robusta than arabica. The peak ofactivity was found at the LG stage for both species. The rapid growthphase of coffee fruit development is between SG and LG stage, a phasethat correlates well with the highest level of SuSy activity. Results inother systems have shown that SuSy activity is highly correlated withsucrose unloading capacity from the phloem at the earliest stages oftomato fruit development (Sun, et al., 1992; Zrenner et al., 1995;N'tchobo, 1999; Wang et al., 1993). The postulate that the level of SuSyactivity indicates the level of sink strength may suggest that the sinkstrength is higher in robusta than arabica. SPS activity has also beendetermined at the same stages of grain maturation. However, SPS activitydid not follow the same schema in robusta and arabica during coffeegrain maturation. While SPS activity fluctuated during robusta graindevelopment, the activity rose steadily during early arabica graindevelopment and stayed at relatively high levels until the end ofmaturation. Without intending to be limited by any explanation ofmechanism, these observations suggest a mechanism in which the steadyincrease in SPS activity seen during arabica grain maturation mayaccount for at least part of the difference in sucrose concentrationfound in mature arabica versus robusta grain. CcSS2 and CcSPS1 mRNAaccumulation in the developing grain was also consistent with theactivity levels detected for the respective enzymes. For SuSy, the levelof CcSS2 transcript accumulation was seen to increase up to the largegreen stage then fall as the grain maturity continued. The level ofCcSPS-C1 transcripts rose consistently, albeit slightly, as arabicagrain maturation progressed, while the level of transcripts was muchlower in the grain of robusta at all stages examined. Again, theexpression data obtained for CcSS2 and CcSPS1 supports the notion thatSPS activity could be the limiting step for re-synthesis of sucroseduring final steps of coffee grain maturation, especially in robustagrain.

In the perspective of genetically improving robusta coffee quality, theSPS enzyme therefore may be a key factor for generating higher finalsucrose concentrations in the mature grain. Alterations in carbonpartitioning in plants, and most particularly improvement of sucroselevels in sink organs, have been accomplished in other plant species.For instance, tomato plants were transformed with a construct comprisinga maize SPS cDNA under the control of the SSU promoter (Rubisco smallsubunit promoter) (Worrell, et al., 1991; Galtier et al. 1993; Foyer andFerrario, 1994; Micallef, et al., 1995; Van Assche et al., 1999;Nguyen-Quoc et al., 1999). The total SPS activity in the leaves of thetransformed plants was six times greater than that of the controls andthe total SPS activity in the mature fruit from the transformed plantswas twice than that of untransformed controls. The combination ofoverexpression, combined with possible de-regulation of enzyme activityin the transgenic tissue were thought to contribute to the overallincrease in SPS activity. The increase in SPS activity was alsoaccompanied by a significant increase (25%) in total overall SuSyactivity in 20 day old tomato fruit. In this case, SuSy activity wasmeasured with an assay in the direction of sucrose breakdown(Nguyen-Quoc et al.; 1999). Fruit from these transgenic tomato linesalso showed higher sugar content (36% increase) compared tountransformed plants (Van Assche et al., 1999). Biochemical studies havealso shown that the high levels of the corn SPS activity in the plantscaused a modification of carbohydrate portioning in the tomato leaveswith an increase of sucrose/starch ratios and also a strong improvementin the photosynthetic capacity. The tomato plants appeared to toleratethe elevated levels of SPS as there were no apparent detrimental growingeffects. In studies by others, plants transformed with the construct35SCaMV-SPS (35 S Cauliflower Mosaïc Virus) were found to have three tofive times more total SPS activity in leaves than in wild-type plants,but tomato fruit obtained from those particular transformants did notshow any increase in SPS activity (Laporte et al. 1997; Nguyen-Quoc etal. 1999). Thus, it appears that the choice of promoter can influencethe ultimate effect of transformation of plants with a heterologous SPSgene.

REFERENCES

-   Altschul S. F., Madden T. L., Schaffer A. A., Zhang J., Zhang Z.,    Miller W. and Lipman D. 1990. Gapped BLAST and PSI-Blast: a new    generation of protein database search. Nucleic Acids Res. 25:    3389-3402.-   Badoud R., 2000. “What do we know about coffee chemistry, flavour    formation and stability? Internal Note, 23 Oct. 2000.-   Bäumlein H, Nagy I, Villarroel R, Inzé D, Wobus U. 1992.    Cis-analysis of a seed protein gene promoter: the conservative RY    repeat CATGCATG within the legumin box is essential for    tissue-specific expression of a legumin gene. Plant J. 2: 233-239.-   BenAmor M. and Mc Carthy J. 2003. Modulation of coffee flavour    precursor levels in green coffee grains. European patent Application    No. 03394056.0 NESTEC S. A.-   Carlson S. J., Chourey P. S., Helentjaris T. and Datta R. 2002. Gene    expression studies on developing kernels of maize sucrose synthase    (SuSy) mutants show evidence for a third SuSy Gene. Plant Mol. Biol.    49: 15-29.-   Chahan Y., Jordon A., Badoud R. and Lindinger W. 2002. From the    green bean to the cup of coffee:investing coffee roasting by on-line    monitoring of volatiles. Eur Food Res Technol. 214:92-104.-   Chourey P. S., Taliercio E. W. and Kane E. J. 1991. Tissue specific    expression and anaerobically induced posttranscriptional modulation    of sucrose synthase genes in Sorghum bicolour M. Plant Physiol.    96:485-490.-   Crouzillat D., Lerceteau E., Petiard V., Morera J., Rodriguez H.,    Walker D., Philips W. R. R., Schnell J., Osei J. and Fritz P. 1996.    Theobroma cacao L.: a genetic linkage map and quantitative trait    loci analysis. Theor Appl Genet. 93: 205-214.-   Echeverria E., Salvucci, M. E., Gonzalez, P., Paris G. and    Salerno G. 1997. Physical and kinetic evidence for an association    between sucrose-phosphate synthase and sucrose-phosphate    phosphatase. Plant Physiol. 115:223-227.-   Foyer C. H. and Ferrario S. 1994. Modulation of carbon and nitrogen    metabolism in transgenic plants with a view to improved biomass    production. In: Lea P J, ed. Transgenic plants and plant    biochemistry. University of Lancaster: Society/Host colloqium,    909-915.-   Fu, H. and Park, W. D. 1995. Sink- and vascular associated sucrose    synthase functions are encoded by different gene classes in potato.    Plant Cell. 7: 1369-1385.-   Galtier N., Foyer C. H., Huber J., Voelker T. A. and    Huber, S. C. 1993. Effects of Elevated Sucrose-Phosphate Synthase    Activity on Photosynthesis, Assimilate Partitioning, and Growth in    Tomato (Lycopersicon esculentum var UC82B). Plant Physiol.    101:535-543.-   Holscher W. and Steinhart H. 1995. Development in Food Science V37A    Food Flavors: Generation, Analysis and Process Influence, Elsevier,    785-803.-   Huang J. W., Chen J. T., Yu W. P., Shyur L. G., Wang A. Y., Sung H.    Y., Lee P. D., and Su J. C. 1996. Complete structures of three rice    sucrose synthase isogenes and differential regulation of their    expressions. Biosci. Biotechnol Biochem. 60: 233-239.-   Huber S. C. and Huber J. L. 1996. Role and regulation of    sucrose-phosphate synthase in higher plants. Annu. Rev. Plant    Physiol. Plant Mol Biol. 47: 431-444.-   Huber S. C., McMichael R. W. Jr, Huber J. L., Bachmann M.,    Yamamoto Y. T. and Conkling M. A. 1995 Light regulation of sucrose    synthsesis: role of protein phosphorylation and possible involvement    of cytosolic Ca²⁺, Carbon Partitioning and Source-Sink Interactions    in Plants, ed. M A Madore, W Lucas, pp. 35-44. Rockville, Md.: Am.    Soc. Plant Physiol.-   Illy, A. and Viani, R. 1995. Espresso Coffee: The Chemistry of    Quality. Academic Press. London Academic Press Ltd.-   Jones T. L. and Ort D. R. 1997. Circadian regulation of sucrose    phosphate synthase activity in tomato by protein phosphatase    activity, Plant Physiol. 113:1167-1175.-   Lafta. A. M. and Lorenzen J. H. 1995. Effect of High Temperature on    Plant Growth and Carbohydrate metabolism in potato. Plant Physiol.    109:637-643.-   Laporte M. M., Galagan J. A., Shapiro J. A., Boersig M. R.,    Shewmaker C. K., Sharkey T. D. 1997. Sucrose-phosphate synthase    activity and yield analysis of tomato plants transformed with maize    sucrose-phosphate synthase. Planta. 203: 253-259.-   Leloup V., Gancel C., Rytz, A. and Pithon, A. 2003. Precursors of    Arabica character in green coffee, chemical and sensory studies. R&D    Report RDOR-RD030009.-   Locher R., Bucheli P. 1998. Comparison of soluble sugar degradation    in soybean seed under simulated tropical storage conditions. Crop    Sci. 38. 1229-1235.-   Lunn J. E. and MacRae E. 2003. New complexities in the synthesis of    sucrose. Curr Opin Plant Biol. 6: 208-214.-   Marraccini P., Deshayes A., Pétiard V. and Rogers W. J. 1999.    Molecular cloning of the complete 11S seed storage protein gene of    Coffea arabica and promoter analysis in the transgenic tobacco    plants. Plant Physiol. Biochem. 37:273-282.-   Marraccini P, Courjault C, Caillet V, Lausanne F, LePage B, Rogers    W, Tessereau S, and Deshayes A. (2003). Rubisco small subunit of    Coffea arabica: cDNA sequence, gene cloning and promoter analysis in    transgenic tobacco plants. Plant Physiol. Biochem. 41:17-25.-   McMichael R. W. Jr, Klein R. R., Salvucci M. E. and Huber S C. 1993.    Identification of the major regulatory phosphorylation site in    sucrose phosphate synthase. Arch. Biochem. Biophys. 321:71-75.-   Micallef, B. J., Haskins, K. A., Vanderveer, P. J., Roh, K.-S.,    Shewmaker, C. K., and Sharkey, T. D. 1995. Altered photosynthesis,    flowering and fruiting in transgenic tomato plants that have an    increased capacity for sucrose synthesis. Planta. 196:327-334.-   N'tchobo H., Dali N., Nguyen-Quoc B., Foyer C. H. and Yelle S. 1999.    Starch synthesis in tomato remains constant throughout fruit    development and is dependent on sucrose supply and sucrose    activity. J. Exp. Bat. 50, 1457-1463.-   Nguyen-Quoc B., N'Tchobo H., Foyer C. H. and Yelle S. 1999.    Overexpression of sucrose-phosphate synthase increases sucrose    unloading in transformed tomato fruit. J. Exp. Bot. 50: 785-791.-   Nguyen-Quoc, B. and C. H. Foyer. 2001. A role for ‘futile cycles’    involving invertase and sucrose synthase in sucrose metabolism of    tomato fruit. J. Exp. Bot. 52:881-889.-   Robinson N. L., Hewitt J. D. and Bennett A. B. 1998. Sink metabolism    in tomato fruit. Plant Physiol. 87:732-730.-   Rogers W. J., Michaux S., Bastin M. and P. Bucheli. 1999. Changes to    the content of sugars, sugar alcohols, myo-inositol, carboxylic    acids and inorganic anions in developing grains from different    varieties of Robusta (Coffea canephora) and Arabica (C. arabica)    coffees. Plant Sc. 149:115-123.-   Russwurm, H. 1969. Fractionation and analysis of aroma precursors in    green coffee, ASIC 4: 103-107.-   Sugden C., Donaghy P. G., Halford N. G., and Hardie D. G. 1999. Two    SNF1-related protein kinases from spinach leaf phosphorylate and    inactivate 3-hydroxy-3-methylglutaryl-coenzyme A reductase, nitrate    reductase, and sucrose phosphate synthase in vitro. Plant Physiol    120:257-274.-   Sun J., Loboda T., Sung S. J. S. and Black, C. C. J. 1992. Sucrose    synthase in wild tomato, Lycopersicon chmielewskii, and tomato fruit    sink strength. Plant Physiol. 98; 1163-1169.-   Toroser D. and Huber S. C. 1997. Protein phosphorylation as a    mechanism for osmotic-stress activation of sucrose-phosphate    synthase in spinach leaves. Plant Physiol. 114:947-955.-   Trevanion S. J., Castleden C. K., Foyer C. H., Furbank R. T.,    Quick W. P. and Lunn J. E. 2004. Regulation of sucrose-phosphate    synthase in wheat (Triticum aestivum) leaves. Functional Plant    Biology. 31:685-695.-   Van Assche, C. Lando, D., Bruneau, J. M., Voelker, T. A.,    Gervais, M. 1999. Modification of sucrose phosphate synthase in    plants. U.S. Pat. No. 5,981,852.-   Wang F., Smith A. G. and Brenner M. L. 1993. Sucrose synthase starch    accumulation and tomato fruit sink strength. Plant Physiol    101:321-327.-   Wang, F., Smith A. G. and Brenner M. L. 1994. Temporal and Spatial    Expression Pattern of Sucrose Synthase during Tomato Fruit    Development. Plant Physiol 104:535-540.-   Worrell, A. C., Bruneau J-M, Summerfelt K., Boersig M. and    Voelker T. A. 1991. Expression of a maize sucrose phosphate synthase    in tomato alters leaf carbohydrate partitioning. Plant Cell    3:1121-1130.-   Zrenner, R., Salanoubat, M., Willmitzer, L., and Sonnewald, U. 1995.    Evidence of crucial role of sucrose synthase for sink strength using    transgenic potato plants (Solanum tuberosum L.). Plant J. 7:97-107.

The present invention is not limited to the embodiments described andexemplified above, but is capable of variation and modification withinthe scope of the appended claims.

What is claimed:
 1. A nucleic acid molecule isolated from coffee (Coffeaspp.) operably linked to a heterologous polynucleotide, the nucleic acidmolecule comprising a coding sequence that encodes a sucrose synthase,wherein the sucrose synthase has an amino acid sequence that (1)comprises one or both of residues 7-554 or 565-727 of SEQ ID NO:8 and(2) is greater than 89% identical to SEQ ID NO:8.
 2. The nucleic acidmolecule of claim 1, wherein the sucrose synthase has an amino acidsequence of SEQ ID NO:8.
 3. The nucleic acid molecule of claim 1,wherein the coding sequence has 90% or greater identity to the codingsequence set forth in SEQ ID NO:
 1. 4. The nucleic acid molecule ofclaim 3, wherein the coding sequence comprises SEQ ID NO:1.
 5. A vectorcomprising the coding sequence of the nucleic acid molecule of claim 1.6. The vector of claim 5, which is an expression vector selected fromthe group of vectors consisting of plasmid, phagemid, cosmid,baculovirus, bacmid, bacterial, yeast and viral vectors.
 7. The vectorof claim 5, wherein the coding sequence of the nucleic acid molecule isoperably linked to a constitutive promoter.
 8. The vector of claim 5,wherein the coding sequence of the nucleic acid molecule is operablylinked to an inducible promoter.
 9. The vector of claim 5, wherein thecoding sequence of the nucleic acid molecule is operably linked to atissue specific promoter.
 10. The vector of claim 9, wherein the tissuespecific promoter is a seed specific promoter.
 11. The vector of claim10, wherein the seed specific promoter is a coffee seed specificpromoter.
 12. A host cell transformed with the vector of claim
 5. 13.The host cell of claim 12, which is a plant cell selected from the groupof plants consisting of coffee, tobacco, Arabidopsis, maize, wheat,rice, soybean barley, rye, oats, sorghum, alfalfa, clover, canola,safflower, sunflower, peanut, cacao, tomatillo, potato, pepper,eggplant, sugar beet, carrot, cucumber, lettuce, pea, aster, begonia,chrysanthemum, delphinium, zinnia, and turfgrasses.
 14. A fertile plantproduced from the plant cell of claim
 13. 15. A method of modulatingflavor or aroma of coffee beans, comprising modulating production oractivity of sucrose synthase within coffee seeds, wherein the sucrosesynthase has an amino acid sequence that (1) comprises one or both ofresidues 7-554 or 565-727 of SEQ ID NO:8 and (2) is greater than 89%identical to SEQ ID NO:8.
 16. The method of claim 15, comprisingincreasing production or activity of the sucrose synthase.
 17. Themethod of claim 16, comprising increasing expression of one or moreendogenous genes encoding sucrose synthase within the coffee seeds. 18.The method of claim 16, comprising introducing a sucrosesynthase-encoding transgene into the plant.
 19. The method of claim 15,comprising decreasing production or activity of the sucrose synthase.20. The method of claim 19, comprising introducing a nucleic acidmolecule into the coffee that inhibits the expression of one or moregenes encoding the sucrose synthase.